Method for making humanized antibodies

ABSTRACT

Variant immunoglobulins, particularly humanized antibody polypeptides are provided, along with methods for their preparation and use. Consensus immunoglobulin sequences and structural models are also provided.

This is a divisional of U.S. Ser. No. 08/146,206 filed Nov. 17, 1993(now U.S. Pat. No. 6,407,213 issued Jun. 18, 2002) which is a 371 ofPCT/US92/05126 filed Jun. 15, 1992 which is a CIP of Ser. No. 07/715,272filed Jun. 14, 1991 (abandoned), the disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods for the preparation and use of variantantibodies and finds application particularly in the fields ofimmunology and cancer diagnosis and therapy.

BACKGROUND OF THE INVENTION

Naturally occurring antibodies (immunoglobulins) comprise two heavychains linked together by disulfide bonds and two light chains, onelight chain being linked to each of the heavy chains by disulfide bonds.Each heavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain(V_(L)) at one end and a constant domain at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light and heavy chain variabledomains, see e.g. Chothia et al., J. Mol. Biol. 186:651-663 (1985);Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985).

The constant domains are not involved directly in binding the antibodyto an antigen, but are involved in various effector functions, such asparticipation of the antibody in antibody-dependent cellularcytotoxicity. The variable domains of each pair of light and heavychains are involved directly in binding the antibody to the antigen. Thedomains of natural light and heavy chains have the same generalstructure, and each domain comprises four framework (FR) regions, whosesequences are somewhat conserved, connected by three hyper-variable orcomplementarity determining regions (CDRs) (see Kabat, E. A. et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., (1987)). The four framework regions largely adopta β-sheet conformation and the CDRs form loops connecting, and in somecases forming part of, the β-sheet structure. The CDRs in each chain areheld in close proximity by the framework regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen bindingsite.

Widespread use has been made of monoclonal antibodies, particularlythose derived from rodents including mice, however they are frequentlyantigenic in human clinical use. For example, a major limitation in theclinical use of rodent monoclonal antibodies is an anti-globulinresponse during therapy (Miller, R. A. et al., Blood 62:988-995 (1983);Schroff, R. W. et al., Cancer Res. 45:879-885 (1985)).

The art has attempted to overcome this problem by constructing“chimeric” antibodies in which an animal antigen-binding variable domainis coupled to a human constant domain (Cabilly et al., U.S. Pat. No.4,816,567; Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA81:6851-6855 (1984); Boulianne, G. L. et al., Nature 312:643-646 (1984);Neuberger, M. S. et al., Nature 314:268-270 (1985)). The term “chimeric”antibody is used herein to describe a polypeptide comprising at leastthe antigen binding portion of an antibody molecule linked to at leastpart of another protein (typically an immunoglobulin constant domain).

The isotype of the human constant domain may be selected to tailor thechimeric antibody for participation in antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (see e.g.Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987); Riechmann, L.et al., Nature 332:323-327 (1988); Love et al., Methods in Enzymology178:515-527 (1989); Bindon, et al., J. Exp. Med. 168:127-142 (1988).

In the typical embodiment, such chimeric antibodies contain about onethird rodent (or other non-human species) sequence and thus are capableof eliciting a significant anti-globulin response in humans. Forexample, in the case of the murine anti-CD3 antibody, OKT3, much of theresulting anti-globulin response is directed against the variable regionrather than the constant region (Jaffers, G. J. et al., Transplantation41:572-578 (1986)).

In a further effort to resolve the antigen binding functions ofantibodies and to minimize the use of heterologous sequences in humanantibodies, Winter and colleagues (Jones, P. T. et al., Nature321:522-525 (1986); Riechmann, L. et al., Nature 332:323-327 (1988);Verhoeyen, M. et al., Science 239:1534-1536 (1988)) have substitutedrodent CDRs or CDR sequences for the corresponding segments of a humanantibody. As used herein, the term “humanized” antibody is an embodimentof chimeric antibodies wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

The therapeutic promise of this approach is supported by the clinicalefficacy of a humanized antibody specific for the CAMPATH-1 antigen withtwo non-Hodgkin lymphoma patients, one of whom had previously developedan anti-globulin response to the parental rat antibody (Riechmann, L. etal., Nature 332:323-327 (1988); Hale, G. et al., Lancet i:1394-1399(1988)). A murine antibody to the interleukin 2 receptor has alsorecently been humanized (Queen, C. et al., Proc. Natl. Acad. Sci. USA86:10029-10033 (1989)) as a potential immunosuppressive reagent.Additional references related to humanization of antibodies include Coet al., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991); Gorman et al.,Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991); Daugherty et al.,Nucleic Acids Research 19(9):2471-2476 (1991); Brown et al., Proc. Natl.Acad. Sci. USA 88:2663-2667 (1991); Junghans et al., Cancer Research50:1495-1502 (1990).

In some cases, substituting CDRs from rodent antibodies for the humanCDRs in human frameworks is sufficient to transfer high antigen bindingaffinity (Jones, P. T. et al., Nature 321:522-525 (1986); Verhoeyen, M.et al., Science 239:1534-1536 (1988)), whereas in other cases it hasbeen necessary to additionally replace one (Riechmann, L. et al., Nature332:323-327 (1988)) or several (Queen, C. et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989)) framework region (FR) residues. See also Coet al., supra.

For a given antibody a small number of FR residues are anticipated to beimportant for antigen binding. Firstly for example, certain antibodieshave been shown to contain a few FR residues which directly contactantigen in crystal structures of antibody-antigen complexes (e.g.,reviewed in Davies, D. R. et al., Ann. Rev. Biochem. 59:439-473 (1990)).Secondly, a number of FR residues have been proposed by Chothia, Leskand colleagues (Chothia, C. & Lesk, A. M., J. Mol. Biol. 196:901-917(1987); Chothia, C. et al., Nature 342:877-883 (1989); Tramontano, A. etal., J. Mol. Biol. 215:175-182 (1990)) as critically affecting theconformation of particular CDRs and thus their contribution to antigenbinding. See also Margolies et al., Proc. Natl. Acad. Sci. USA72:2180-2184 (1975).

It is also known that, in a few instances, an antibody variable domain(either V_(H) or V_(L)) may contain glycosylation sites, and that thisglycosylation may improve or abolish antigen binding, Pluckthun,Biotechnology 9:545-51 (1991); Spiegelberg et al., Biochemistry9:4217-4223 (1970); Wallic et al, J. Exp. Med. 168:1099-1109 (1988); Soxet al., Proc. Natl. Acad. Sci. USA 66:975-982 (1970); Margni et al.,Ann. Rev. Immunol. 6:535-554 (1988). Ordinarily, however, glycosylationhas no influence on the antigen-binding properties of an antibody,Pluckthun, supra, (1991).

The three-dimensional structure of immunoglobulin chains has beenstudied, and crystal structures for intact immunoglobulins, for avariety of immunoglobulin fragments, and for antibody-antigen complexeshave been published (see e.g., Saul et al., Journal of BiologicalChemistry 25:585-97 (1978); Sheriff et al., Proc. Natl. Acad. Sci. USA84:8075-79 (1987); Segal et al., Proc. Natl. Acad. Sci. USA 71:4298-4302(1974); Epp et al., Biochemistry 14(22):49434952 (1975); Marquart etal., J. Mol. Biol. 141:369-391 (1980); Furey et al., J. Mol. Biol.167:661-692 (1983); Snow and Amzel, Protein: Structure, Function, andGenetics 1:267-279, Alan R. Liss, Inc. pubs. (1986); Chothia and Lesk,J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:877-883(1989); Chothia et al., Science 233:755-58 (1986); Huber et al., Nature264:415420 (1976); Bruccoleri et al., Nature 335:564-568 (1988) andNature 336:266 (1988); Sherman et al., Journal of Biological Chemistry263:4064-4074 (1988); Amzel and Poijak, Ann. Rev. Biochem. 48:961-67(1979); Silverton et al., Proc. Natl. Acad. Sci. USA 74:5140-5144(1977); and Gregory et al., Molecular Immunology 24:821-829 (1987). Itis known that the function of an antibody is dependent on its threedimensional structure, and that amino acid substitutions can change thethree-dimensional structure of an antibody, Snow and Amzel, supra. Ithas previously been shown that the antigen binding affinity of ahumanized antibody can be increased by mutagenesis based upon molecularmodelling (Riechmann, L. et al., Nature 332:323-327 (1988); Queen, C. etal., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)).

Humanizing an antibody with retention of high affinity for antigen andother desired biological activities is at present difficult to achieveusing currently available procedures. Methods are needed forrationalizing the selection of sites for substitution in preparing suchantibodies and thereby increasing the efficiency of antibodyhumanization.

The proto-oncogene HER2 (human epidermal growth factor receptor 2)encodes a protein tyrosine kinase (p185^(HER2)) that is related to andsomewhat homologous to the human epidermal growth factor receptor (seeCoussens, L. et al., Science 230:1132-1139 (1985); Yamamoto, T. et al.,Nature 319:230-234 (1986); King, C. R. et al., Science 229:974-976(1985)). HER2 is also known in the field as c-erbB-2, and sometimes bythe name of the rat homolog, neu. Amplification and/or overexpression ofHER2 is associated with multiple human malignancies and appears to beintegrally involved in progression of 25-30% of human breast and ovariancancers (Slamon, D. J. et al., Science 235:177-182 (1987), Slamon, D. J.et al., Science 244:707-712 (1989)). Furthermore, the extent ofamplification is inversely correlated with the observed median patientsurvival time (Slamon, supra, Science 1989).

The murine monoclonal antibody known as muMAb4D5 (Fendly, B. M. et al.,Cancer Res. 50:1550-1558 (1990)), directed against the extracellulardomain (ECD) of p185^(HER2), specifically inhibits the growth of tumorcell lines overexpressing p185^(HER2) in monolayer culture or in softagar (Hudziak, R. M. et al., Molec. Cell. Biol. 9:1165-1172 (1989);Lupu, R. et al, Science 249:1552-1555 (1990)). MuMAb4D5 also has thepotential of enhancing tumor cell sensitivity to tumor necrosis factor,an important effector molecule in macrophage-mediated tumor cellcytotoxicity (Hudziak, supra, 1989; Shepard, H. M. and Lewis, G. D. J.Clinical Immunology 8:333-395 (1988)). Thus muMAb4D5 has potential forclinical intervention in and imaging of carcinomas in which p185^(HER2)is overexpressed. The muMAb4D5 and its uses are described in PCTapplication WO 89/06692 published 27 Jul. 1989. This murine antibody wasdeposited with the ATCC and designated ATCC CRL 10463. However, thisantibody may be immunogenic in humans.

It is therefore an object of this invention to provide methods for thepreparation of antibodies which are less antigenic in humans thannon-human antibodies but have desired antigen binding and othercharacteristics and activities.

It is a further object of this invention to provide methods for theefficient humanization of antibodies, i.e. selecting non-human aminoacid residues for importation into a human antibody background sequencein such a fashion as to retain or improve the affinity of the non-humandonor antibody for a given antigen.

It is another object of this invention to provide humanized antibodiescapable of binding p185^(HER2).

Other objects, features, and characteristics of the present inventionwill become apparent upon consideration of the following description andthe appended claims.

SUMMARY OF THE INVENTION

The objects of this invention are accomplished by a method for making ahumanized antibody comprising amino acid sequence of an import,non-human antibody and a human antibody, comprising the steps of:

a. obtaining the amino acid sequences of at least a portion of an importantibody variable domain and of a consensus variable domain;

b. identifying Complementarity Determining Region (CDR) amino acidsequences in the import and the human variable domain sequences;

c. substituting an import CDR amino acid sequence for the correspondinghuman CDR amino acid sequence;

d. aligning the amino acid sequences of a Framework Region (FR) of theimport antibody and the corresponding FR of the consensus antibody;

e. identifying import antibody FR residues in the aligned FR sequencesthat are non-homologous to the corresponding consensus antibodyresidues;

f. determining if the non-homologous import amino acid residue isreasonably expected to have at least one of the following effects:

1. non-covalently binds antigen directly,

2. interacts with a CDR; or

3. participates in the V_(L)-V_(H) interface; and

g. for any non-homologous import antibody amino acid residue which isreasonably expected to have at least one of these effects, substitutingthat residue for the corresponding amino acid residue in the consensusantibody FR sequence.

Optionally, the method of this invention comprises the additional stepsof determining if any non-homologous residues identified in step (e) areexposed on the surface of the domain or buried within it, and if theresidue is exposed but has none of the effects identified in step (f),retaining the consensus residue.

Additionally, in certain embodiments the method of this inventioncomprises the feature wherein the corresponding consensus antibodyresidues identified in step (e) above are selected from the groupconsisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L,66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H,39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 76H,78H, 91H, 92H, 93H, and 103H (utilizing the numbering system set forthin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987)).

In certain embodiments, the method of this invention comprises theadditional steps of searching either or both of the import, non-humanand the consensus variable domain sequences for glycosylation sites,determining if the glycosylation is reasonably expected to be importantfor the desired antigen binding and biological activity of the antibody(i.e., determining if the glycosylation site binds to antigen or changesa side chain of an amino acid residue that binds to antigen, or if theglycosylation enhances or weakens antigen binding, or is important formaintaining antibody affinity). If the import sequence bears theglycosylation site, it is preferred to substitute that site for thecorresponding residues in the consensus human if the glycosylation siteis reasonably expected to be important. If only the consensus sequence,and not the import, bears the glycosylation site, it is preferred toeliminate that glycosylation site or substitute therefor thecorresponding amino acid residues from the import sequence.

Another embodiment of this invention comprises aligning import antibodyand the consensus antibody FR sequences, identifying import antibody FRresidues which are non-homologous with the aligned consensus FRsequence, and for each such non-homologous import antibody FR residue,determining if the corresponding consensus antibody residue represents aresidue which is highly conserved across all species at that site, andif it is so conserved, preparing a humanized antibody which comprisesthe consensus antibody amino acid residue at that site.

Certain alternate embodiments of the methods of this invention compriseobtaining the amino acid sequence of at least a portion of an import,non-human antibody variable domain having a CDR and a FR, obtaining theamino acid sequence of at least a portion of a consensus antibodyvariable domain having a CDR and a FR, substituting the non-human CDRfor the human CDR in the consensus antibody variable domain, and thensubstituting an amino acid residue for the consensus amino acid residueat at least one of the following sites:

a. (in the FR of the variable domain of the light chain) 4L, 35L, 36L,38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L,71L, 73L, 85L, 87L, 98L, or

b. (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H,36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H,75H, 76H, 78H, 91H, 92H, 93H, and 103H.

In preferred embodiments, the non-CDR residue substituted at theconsensus FR site is the residue found at the corresponding location ofthe non-human antibody.

Optionally, this just-recited embodiment comprises the additional stepsof following the method steps appearing at the beginning of this summaryand determining whether a particular amino acid residue can reasonablybe expected to have undesirable effects.

This invention also relates to a humanized antibody comprising the CDRsequence of an import, non-human antibody and the FR sequence of a humanantibody, wherein an amino acid residue within the human FR sequencelocated at any one of the sites 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L,62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L,2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H,73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H has been substituted byanother residue. In preferred embodiments, the residue substituted atthe human FR site is the residue found at the corresponding location ofthe non-human antibody from which the non-human CDR was obtained. Inother embodiments, no human FR residue other than those set forth inthis group has been substituted.

This invention also encompasses specific humanized antibody variabledomains, and isolated polypeptides having homology with the followingsequences.

1. SEQ. ID NO. 1, which is the light chain variable domain of ahumanized version of muMAb4D5:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT

2. SEQ. ID NO. 2, which is the heavy chain variable domain of ahumanized version of muMAb4D5):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLV TVSS

In another aspect, this invention provides a consensus antibody variabledomain amino acid sequence for use in the preparation of humanizedantibodies, methods for obtaining, using, and storing a computerrepresentation of such a consensus sequence, and computers comprisingthe sequence data of such a sequence. In one embodiment, the followingconsensus antibody variable domain amino acid sequences are provided:

SEQ. ID NO. 3 (light chain):

DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT, and

SEQ. ID NO. 4 (heavy chain):

EVQLVESGGGLVOPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTL VTVSS

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the comparison of the V_(L) domain amino acid residues ofmuMAb4D5, huMAb4D5, and a consensus sequence (FIG. 1A, SEQ. ID NO. 5,SEQ. ID NO. 1 and SEQ. ID NO. 3, respectively). FIG. 1B shows thecomparison between the V_(H) domain amino acid residues nuMAb4D5,huMAb4D5, and a consensus sequence (FIG. 1B, SEQ. ID NO. 6, SEQ. ID NO.2 and SEQ. ID NO. 4, respectively). Both FIGS. 1A and 1B use thegenerally accepted numbering scheme from Kabat, E. A., et al, Sequencesof Proteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987)). In both FIG. 1A and FIG. 1B, the CDR residuesdetermined according to a standard sequence definition (as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987)) are indicated by the firstunderlining beneath the sequences, and the CDR residues determinedaccording to a structural definition (as in Chothia, C. & Lesk, A. M.,J. Mol. Biol. 196:901-917 (1987)) are indicated by the second, lowerunderlines. The mismatches between genes are shown by the verticallines.

FIG. 2 shows a scheme for humanization of muMAb4D5 V_(L) and V_(H) bygene conversion mutagenesis.

FIG. 3 shows the inhibition of SK-BR-3 proliferation by MAb4D5 variants.Relative cell proliferation was determined as described (Hudziak, R. M.et al., Molec. Cell. Biol. 9:1165-1172 (1989)) and data (average oftriplicate determinations) are presented as a percentage of results withuntreated cultures for muMAb4D5, huMAb4D5-8 (∘) and huMAb4D5-1 (□).

FIG. 4 shows a stereo view of α-carbon tracing for a model of huMAb4D5-8V_(L) and V_(H). The CDR residues (Kabat, E. A. et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)) are shown in bold and side chains of V_(H)residues A71, T73, A78, S93, Y102 and V_(L) residues Y55 plus R66 (seeTable 3) are shown.

FIG. 5 shows an amino acid sequence comparison of V_(L) (top panel) andV_(H) (lower panel) domains of the murine anti-CD3 monoclonal Ab UCHT1(muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992) with ahumanized variant of this antibody (huxCD3v1). Also shown are consensussequences (most commonly occurring residue or pair of residues) of themost abundant human subgroups, namely V_(L) κ 1 and V_(H) III upon whichthe humanized sequences are based (Kabat, E. A. et al., Sequences ofProteins of Immunological Interest, 5^(th) edition, National Institutesof Health, Bethesda, Md., USA (1991)). The light chain sequences—muxCD3,huxCD3v1 and huκI—correspond to SEQ.ID.NOs 16, 17, and 18, respectively.The heavy chain sequences—muxCD3, huxCD3v1 and huIII—correspond to SEQ.ID. NOs 19,26, and 21, respectively. Residues which differ betweenmuxCD3 and huxCD3v1 are identified by an asterisk (*), whereas thosewhich differ between humanized and consensus sequences are identified bya sharp sign (#). A bullet (•) denotes that a residue at this positionhas been found to contact antigen in one or more crystallographicstructures of antibody/antigen complexes (Kabat et al., 1991; Mian, I.S. et al., J. Mol. Biol. 217, 133-151 (1991)). The location of CDRresidues according to a sequence definition (Kabat et al., 1991) and astructural definition (Chothia and Lesk, supra 1987) are shown by a lineand carats ({circumflex over ( )}) beneath the sequences, respectively.

FIGS. 6A-1 and 6A-2 compare compares murine and humanized amino acidsequences for the heavy chain of an anti-CD18 antibody. H52H4-160 (SEQ.ID. NO. 22) is the murine sequence, and pH52-8.0 (SEQ. ID. NO. 23) isthe humanized heavy chain sequence. pH52-8.0 residue 143S is the finalamino acid in the variable heavy chain domain V_(H), and residue 144A isthe first amino acid in the constant heavy chain domain C_(H1).

FIG. 6B compares murine and humanized amino acid sequences for the lightchain of an anti-CD18 antibody. H52L6-158 (SEQ. ID. NO. 24) is themurine sequence, and pH52-9.0 (SEQ. ID. NO. 25) is the humanized lightchain sequence. pH52-9.0 residue 128T is the final amino acid in thelight chain variable domain V_(L), and residue 129V is the first aminoacid in the light chain constant domain C_(L).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In general, the following words or phrases have the indicateddefinitions when used in the description, examples, and claims:

The murine monoclonal antibody known as muMAb4D5 (Fendly, B. M. et al.,Cancer Res. 50:1550-1558 (1990)) is directed against the extracellulardomain (ECD) of p185^(HER2) The muMAb4D5 and its uses are described inPCT application WO 89/06692 published 27 Jul. 1989. This murine antibodywas deposited with the ATCC and designated ATCC CRL 10463. In thisdescription and claims, the terms muMAb4D5, chMAb4D5 and huMAb4D5represent murine, chimerized and humanized versions of the monoclonalantibody 4D5, respectively.

A humanized antibody for the purposes herein is an immunoglobulin aminoacid sequence variant or fragment thereof which is capable of binding toa predetermined antigen and which, comprises a FR region havingsubstantially the amino acid sequence of a human immunoglobulin and aCDR having substantially the amino acid sequence of a non-humanimmunoglobulin.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are referred to herein as “import” residues, whichare typically taken from an “import” antibody domain, particularly avariable domain. An import residue, sequence, or antibody has a desiredaffinity and/or specificity, or other desirable antibody biologicalactivity as discussed herein.

In general the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains (Fab, Fab′, F(ab′)₂,Fabc, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. Ordinarily, the antibody will containboth the light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain.

The humanized antibody will be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is acomplement fixing constant domain where it is desired that the humanizedantibody exhibit cytotoxic activity, and the class is typically IgG₁.Where such cytotoxic activity is not desirable, the constant domain maybe of the IgG₂ class. The humanized antibody may comprise sequences frommore than one class or isotype, and selecting particular constantdomains to optimize desired effector functions is within the ordinaryskill in the art.

The FR and CDR regions of the humanized antibody need not correspondprecisely to the parental sequences, e.g., the import CDR or theconsensus FR may be mutagenized by substitution, insertion or deletionof at least one residue so that the CDR or FR residue at that site doesnot correspond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parental FRand CDR sequences, more often 90%, and most preferably greater than 95%.

In general, humanized antibodies prepared by the method of thisinvention are produced by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen.

Residues that influence antigen binding are defined to be residues thatare substantially responsible for the antigen affinity or antigenspecificity of a candidate immunoglobulin, in a positive or a negativesense. The invention is directed to the selection and combination of FRresidues from the consensus and import sequence so that the desiredimmunoglobulin characteristic is achieved. Such desired characteristicsinclude increases in affinity and greater specificity for the targetantigen, although it is conceivable that in some circumstances theopposite effects might be desired. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding(although not all CDR residues are so involved and therefore need not besubstituted into the consensus sequence). However, FR residues also havea significant effect and can exert their influence in at least threeways: They may noncovalently directly bind to antigen, they may interactwith CDR residues and they may affect the interface between the heavyand light chains.

A residue that noncovalently directly binds to antigen is one that, bythree dimensional analysis, is reasonably expected to noncovalentlydirectly bind to antigen. Typically, it is necessary to impute theposition of antigen from the spatial location of neighboring CDRs andthe dimensions and structure of the target antigen. In general, onlythose humanized antibody residues that are capable of forming saltbridges, hydrogen bonds, or hydrophobic interactions are likely to beinvolved in non-covalent antigen binding, however residues which haveatoms which are separated from antigen spatially by 3.2 Angstroms orless may also non-covalently interact with antigen. Such residuestypically are the relatively larger amino acids having the side chainswith the greatest bulk, such as tyrosine, arginine, and lysine.Antigen-binding FR residues also typically will have side chains thatare oriented into an envelope surrounding the solvent oriented face of aCDR which extends about 7 Angstroms into the solvent from the CDR domainand about 7 Angstroms on either side of the CDR domain, again asvisualized by three dimensional modeling.

A residue that interacts with a CDR generally is a residue that eitheraffects the conformation of the CDR polypeptide backbone or forms anoncovalent bond with a CDR residue side chain. Conformation-affectingresidues ordinarily are those that change the spatial position of anyCDR backbone atom (N, Cα, C, O, Cβ) by more than about 0.2 Angstroms.Backbone atoms of CDR sequences are displaced for example by residuesthat interrupt or modify organized structures such as beta sheets,helices or loops. Residues that can exert a profound affect on theconformation of neighboring sequences include proline and glycine, bothof which are capable of introducing bends into the backbone. Otherresidues that can displace backbone atoms are those that are capable ofparticipating in salt bridges and hydrogen bonds.

A residue that interacts with a CDR side chain is one that is reasonablyexpected to form a noncovalent bond with a CDR side chain, generallyeither a salt bridge or hydrogen bond. Such residues are identified bythree dimensional positioning of their side chains. A salt or ion bridgecould be expected to form between two side chains positioned withinabout 2.5-3.2 Angstroms of one another that bear opposite charges, forexample a lysinyl and a glutamyl pairing. A hydrogen bond could beexpected to form between the side chains of residue pairs such as serylor threonyl with aspartyl or glutamyl (or other hydrogen acceptingresidues). Such pairings are well known in the protein chemistry art andwill be apparent to the artisan upon three dimensional modeling of thecandidate immunoglobulin.

Immunoglobulin residues that affect the interface between heavy andlight chain variable regions (“the V_(L)-V_(H) interface”) are thosethat affect the proximity or orientation of the two chains with respectto one another. Certain residues involved in interchain interactions arealready known and include V_(L) residues 34, 36, 38, 44, 46, 87, 89, 91,96, and 98 and V_(H) residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and103 (utilizing the nomenclature set forth in Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)). Additional residues are newly identified by theinventors herein, and include 43L, 85L, 43H and 60H. While theseresidues are indicated for IgG only, they are applicable across species.In the practice of this invention, import antibody residues that arereasonably expected to be involved in interchain interactions areselected for substitution into the consensus sequence. It is believedthat heretofore no humanized antibody has been prepared with anintrachain-affecting residue selected from an import antibody sequence.

Since it is not entirely possible to predict in advance what the exactimpact of a given substitution will be it may be necessary to make thesubstitution and assay the candidate antibody for the desiredcharacteristic. These steps, however, are per se routine and well withinthe ordinary skill of the art.

CDR and FR residues are determined according to a standard sequencedefinition (Kabat et al., Sequences of Proteins of ImmunologicalInterest, National Institutes of Health, Bethesda Md. (1987), and astructural definition (as in Chothia and Lesk, J. Mol. Biol. 196:901-917(1987). Where these two methods result in slightly differentidentifications of a CDR, the structural definition is preferred, butthe residues identified by the sequence definition method are consideredimportant FR residues for determination of which framework residues toimport into a consensus sequence.

Throughout this description, reference is made to the numbering schemefrom Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987) and(1991). In these compendiums, Kabat lists many amino acid sequences forantibodies for each subclass, and lists the most commonly occurringamino acid for each residue position in that subclass. Kabat uses amethod for assigning a residue number to each amino acid in a listedsequence, and this method for assigning residue numbers has becomestandard in the field. The Kabat numbering scheme is followed in thisdescription.

For purposes of this invention, to assign residue numbers to a candidateantibody amino acid sequence which is not included in the Kabatcompendium, one follows the following steps. Generally, the candidatesequence is aligned with any immunoglobulin sequence or any consensussequence in Kabat. Alignment may be done by hand, or by computer usingcommonly accepted computer programs; an example of such a program is theAlign 2 program discussed in this description. Alignment may befacilitated by using some amino acid residues which are common to mostFab sequences. For example, the light and heavy chains each typicallyhave two cysteines which have the same residue numbers; in V_(L) domainthe two cysteines are typically at residue numbers 23 and 88, and in theV_(H) domain the two cysteine residues are typically numbered 22 and 92.Framework residues generally, but not always, have approximately thesame number of residues, however the CDRs will vary in size. Forexample, in the case of a CDR from a candidate sequence which is longerthan the CDR in the sequence in Kabat to which it is aligned, typicallysuffixes are added to the residue number to indicate the insertion ofadditional residues (see, e.g. residues 100abcde in FIG. 5). Forcandidate sequences which, for example, align with a Kabat sequence forresidues 34 and 36 but have no residue between them to align withresidue 35, the number 35 is simply not assigned to a residue.

Thus, in humanization of an import variable sequence, where one cuts outan entire human or consensus CDR and replaces it with an import CDRsequence, (a) the exact number of residues may be swapped, leaving thenumbering the same, (b) fewer import amino acid residues may beintroduced than are cut, in which case there will be a gap in theresidue numbers, or (c) a larger number of amino acid residues may beintroduced then were cut, in which case the numbering will involve theuse of suffixes such as 100abcde.

The terms “consensus sequence” and “consensus antibody” as used hereinrefers to an amino acid sequence which comprises the most frequentlyoccurring amino acid residues at each location in all immunoglobulins ofany particular subclass or subunit structure. The consensus sequence maybe based on immunoglobulins of a particular species or of many species.A “consensus” sequence, structure, or antibody is understood toencompass a consensus human sequence as described in certain embodimentsof this invention, and to refer to an amino acid sequence whichcomprises the most frequently occurring amino acid residues at eachlocation in all human immunoglobulins of any particular subclass orsubunit structure. This invention provides consensus human structuresand consensus structures which consider other species in addition tohuman.

The subunit structures of the five immunoglobulin classes in humans areas follows:

Class Heavy Chain Subclasses Light Chain Molecular Formula IgG γ γ1, γ2,κ or λ (γ₂κ₂), (γ₂λ₂) γ3, γ4 IgA α α1, α2 κ or λ (α₂κ₂)_(n) ^(*),(α₂λ₂)_(n) ^(*) IgM μ none κ or λ (μ₂κ₂)₅, (μ₂λ₂)₅ IgD δ none κ or λ(δ₂κ₂), (δ₂λ₂) IgE ε none κ or λ (ε₂κ₂), (ε₂λ₂) (*_(n) may equal 1, 2,or 3)

In preferred embodiments of an IgGγ1 human consensus sequence, theconsensus variable domain sequences are derived from the most abundantsubclasses in the sequence compilation of Kabat et al., Sequences ofProteins of Immunological Interest, National Institutes of Health,Bethesda Md. (1987), namely V_(L) κ subgroup I and V_(H) group III. Insuch preferred embodiments, the V_(L) consensus domain has the aminoacid sequence:

DIQMTQSPSSLSASVGDRVTITCRASODVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ. ID NO. 3);

the V_(H) consensus domain has the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ. ID NO.4).

These sequences include consensus CDRs as well as consensus FR residues(see for example in FIG. 1).

While not wishing to be limited to any particular theories, it may bethat these preferred embodiments are less likely to be immunogenic in anindividual than less abundant subclasses. However, in other embodiments,the consensus sequence is derived from other subclasses of humanimmunoglobulin variable domains. In yet other embodiments, the consensussequence is derived from human constant domains.

Identity or homology with respect to a specified amino acid sequence ofthis invention is defined herein as the percentage of amino acidresidues in a candidate sequence that are identical with the specifiedresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal or internal extensions, deletions, or insertionsinto the specified sequence shall be construed as affecting homology.All sequence alignments called for in this invention are such maximalhomology alignments. While such alignments may be done by hand usingconventional methods, a suitable computer program is the “Align 2”program for which protection is being sought from the U.S. Register ofCopyrights (Align 2, by Genentech, Inc., application filed Dec. 9,1991).

“Non-homologous” import antibody residues are those residues which arenot identical to the amino acid residue at the analogous orcorresponding location in a consensus sequence, after the import andconsensus sequences are aligned.

The term “computer representation” refers to information which is in aform that can be manipulated by a computer. The act of storing acomputer representation refers to the act of placing the information ina form suitable for manipulation by a computer.

This invention is also directed to novel polypeptides, and in certainaspects, isolated novel humanized anti-p185^(HER2) antibodies areprovided. These novel anti-p185^(HER2) antibodies are sometimescollectively referred to herein as huMAb4D5, and also sometimes as thelight or heavy chain variable domains of huMAb4D5, and are definedherein to be any polypeptide sequence which possesses a biologicalproperty of a polypeptide comprising the following polypeptide sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT (SEQ. ID NO. 1, whichis the light chain variable domain of huMAb4D5); or

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLV TVSS (SEQ. IDNO. 2, which is the heavy chain variable domain of huMAb4D5).

“Biological property”, as relates for example to anti-p185^(HER2), forthe purposes herein means an in vivo effector or antigen-bindingfunction or activity that is directly or indirectly performed byhuMAb4D5 (whether in its native or denatured conformation). Effectorfunctions include p185^(HER2) binding, any hormonal or hormonalantagonist activity, any mitogenic or agonist or antagonist activity,any cytotoxic activity. An antigenic function means possession of anepitope or antigenic site that is capable of cross-reacting withantibodies raised against the polypeptide sequence of huMAb4D5.

Biologically active huMAb4D5 is defined herein as a polypeptide thatshares an effector function of huMAb4D5. A principal known effectorfunction of huMAb4D5 is its ability to bind to p185^(HER2).

Thus, the biologically active and antigenically active huMAb4D5polypeptides that are the subject of certain embodiments of thisinvention include the sequence of the entire translated nucleotidesequence of huMAb4D5; mature huMAb4D5; fragments thereof having aconsecutive sequence of at least 5, 10, 15, 20,25, 30 or 40 amino acidresidues comprising sequences from muMAb4D5 plus residues from the humanFR of huMAb4D5; amino acid sequence variants of huMAb4D5 wherein anamino acid residue has been inserted N- or C-terminal to, or within,huMAb4D5 or its fragment as defined above; amino acid sequence variantsof huMAb4D5 or its fragment as defined above wherein an amino acidresidue of huMAb4D5 or its fragment as defined above has beensubstituted by another residue, including predetermined mutations by,e.g., site-directed or PCR mutagenesis; derivatives of huMAb4D5 or itsfragments as defined above wherein huMAb4D5 or its fragments have beencovalent modified, by substitution, chemical, enzymatic, or otherappropriate means, with a moiety other than a naturally occurring aminoacid; and glycosylation variants of huMAb4D5 (insertion of aglycosylation site or deletion of any glycosylation site by deletion,insertion or substitution of suitable residues). Such fragments andvariants exclude any polypeptide heretofore identified, includingmuMAb4D5 or any known polypeptide fragment, which are anticipatory order35 U.S.C. 102 as well as polypeptides obvious thereover under 35 U.S.C.103.

An “isolated” polypeptide means polypeptide which has been identifiedand separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with diagnostic or therapeutic uses forthe polypeptide, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, forexample, a polypeptide product comprising huMAb4D5 will be purified froma cell culture or other synthetic environment (1) to greater than 95% byweight of protein as determined by the Lowry method, and most preferablymore than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of agas- or liquid-phase sequenator (such as a commercially availableApplied Biosystems sequenator Model 470, 477, or 473), or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or, preferably, silver stain. Isolated huMAb4D5 includeshuMAb4D5 in situ within recombinant cells since at least one componentof the huMAb4D5 natural environment will not be present. Ordinarily,however, isolated huMAb4D5 will be prepared by at least one purificationstep.

In accordance with this invention, huMAb4D5 nucleic acid is RNA or DNAcontaining greater than ten bases that encodes a biologically orantigenically active huMAb4D5, is complementary to nucleic acid sequenceencoding such huMAb4D5, or hybridizes to nucleic acid sequence encodingsuch huMAb4D5 and remains stably bound to it under stringent conditions,and comprises nucleic acid from a muMAb4D5 CDR and a human FR region.

Preferably, the huMAb4D5 nucleic acid encodes a polypeptide sharing atleast 75% sequence identity, more preferably at least 80%, still morepreferably at least 85%, even more preferably at 90%, and mostpreferably 95%, with the huMAb4D5 amino acid sequence. Preferably, anucleic acid molecule that hybridizes to the huMAb4D5 nucleic acidcontains at least 20, more preferably 40, and most preferably 90 bases.Such hybridizing or complementary nucleic acid, however, is furtherdefined as being novel under 35 U.S.C. 102 and unobvious under 35 U.S.C.103 over any prior art nucleic acid.

Stringent conditions are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0/1% NaDodSO₄ at 50° C.; (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0/1% FicoII/0/1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in0.2×SSC and 0.1% SDS.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Howeverenhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

An “exogenous” element is defined herein to mean nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032 published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14: 5399-5407 [1986]). They are thenpurified on polyacrylamide gels.

The technique of “polymerase chain reaction,” or “PCR,” as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA and/or DNA, are amplified as described inU.S. Pat. No. 4,683,195 issued 28 Jul. 1987. Generally, sequenceinformation from the ends of the region of interest or beyond needs tobe available, such that oligonucleotide primers can be designed; theseprimers will be identical or similar in sequence to opposite strands ofthe template to be amplified. The 5′ terminal nucleotides of the twoprimers may coincide with the ends of the amplified material. PCR can beused to amplify specific RNA sequences, specific DNA sequences fromtotal genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. See generally Mullis et al.,Cold Spring Harbor Symp. Ouant. Biol., 51: 263 (1987); Erlich, ed., PCRTechnology. (Stockton Press, NY, 1989). As used herein, PCR isconsidered to be one, but not the only, example of a nucleic acidpolymerase reaction method for amplifying a nucleic acid test sample,comprising the use of a known nucleic acid (DNA or RNA) as a primer andutilizes a nucleic acid polymerase to amplify or generate a specificpiece of nucleic acid or to amplify or generate a specific piece ofnucleic acid which is complementary to a particular nucleic acid.

Suitable Methods for Practicing the Invention

Some aspects of this invention include obtaining an import, non-humanantibody variable domain, producing a desired humanized antibodysequence and for humanizing an antibody gene sequence are describedbelow. A particularly preferred method of changing a gene sequence, suchas gene conversion from a non-human or consensus sequence into ahumanized nucleic acid sequence, is the cassette mutagenesis proceduredescribed in Example 1. Additionally, methods are given for obtainingand producing antibodies generally, which apply equally to nativenon-human antibodies as well as to humanized antibodies.

Generally, the antibodies and antibody variable domains of thisinvention are conventionally prepared in recombinant cell culture, asdescribed in more detail below. Recombinant synthesis is preferred forreasons of safety and economy, but it is known to prepare peptides bychemical synthesis and to purify them from natural sources; suchpreparations are included within the definition of antibodies herein.

Molecular Modeling

An integral step in our approach to antibody humanization isconstruction of computer graphics models of the import and humanizedantibodies. These models are used to determine if the sixcomplementarity-determining regions (CDRs) can be successfullytransplanted from the import framework to a human one and to determinewhich framework residues from the import antibody, if any, need to beincorporated into the humanized antibody in order to maintain CDRconformation. In addition, analysis of the sequences of the import andhumanized antibodies and reference to the models can help to discernwhich framework residues are unusual and thereby might be involved inantigen binding or maintenance of proper antibody structure.

All of the humanized antibody models of this invention are based on asingle three-dimensional computer graphics structure hereafter referredto as the consensus structure. This consensus structure is a keydistinction from the approach of previous workers in the field, whotypically begin by selecting a human antibody structure which has anamino acid sequence which is similar to the sequence of their importantibody.

The consensus structure of one embodiment of this invention was built infive steps as described below.

Step 1: Seven Fab X-ray crystal structures from the Brookhaven ProteinData Bank were used (entries 2FB4, 2RHE, 3FAB, and 1REI which are humanstructures, and 2MCP, 1FBJ, and 2HFL which are murine structures). Foreach structure, protein mainchain geometry and hydrogen bonding patternswere used to assign each residue to one of three secondary structuretypes: alpha-helix, beta-strand or other (i.e. non-helix andnon-strand). The immunoglobulin residues used in superpositioning andthose included in the consensus structure are shown in Table 1.

TABLE 1 Immunoglobulin Residues Used in Superpositioning and ThoseIncluded in the Consensus Structure V_(L)κ domain Ig^(a) 2FB4 2RHE 2MCP3FAB 1FBJ 2HFL 1REI Consensus^(b)  2-11 18-24 18-24 19-25 18-24 19-2519-25 19-25 16-27 32-37 34-39 39-44 32-37 32-37 32-37 33-38 33-39 41-4960-66 62-68 67-72 53-66 60-65 60-65 61-66 59-77 69-74 71-76 76-81 69-7469-74 69-74 70-75 84-88 86-90 91-95 84-88 84-88 84-88 85-89 82-91101-105 RMS^(c) 0.40 0.60 0.53 0.54 0.48 0.50 V_(H) domain Ig^(a) 2FB42MCP 3FAB 1FBJ 2HFL Consensus^(b) 3-8 18-25 18-25 18-25 18-25 18-2517-23 34-39 34-39 34-39 34-39 34-39 33-41 46-52 46-52 46-52 46-52 46-5245-51 57-61 59-63 56-60 57-61 57-61 57-61 68-71 70-73 67-70 68-71 68-7166-71 78-84 80-86 77-83 78-84 78-84 75-82 92-99 94-101 91-98 92-99 92-9988-94 102-108 RMS^(c) 0.43 0.85 0.62 0.91 RMS^(d) 0.91 0.73 0.77 0.92^(a)Four-letter code for Protein Data Bank file. ^(b)Residue numbers forthe crystal structures are taken from the Protein Data Bank files.Residue numbers for the consensus structure are according to Kabat etal. ^(c)Root-mean-square deviation in Å for (N, Ca, C) atomssuperimposed on 2FB4. ^(d)Root-mean-square deviation in Å for (N, Ca, C)atoms superimposed on 2HFL.

Step 2: Having identified the alpha-helices and beta-strands in each ofthe seven structures, the structures were superimposed on one anotherusing the INSIGHT computer program (Biosym Technologies, San Diego,Calif.) as follows: The 2FB4 structure was arbitrarily chosen as thetemplate (or reference) structure. The 2FB4 was held fixed in space andthe other six structures rotated and translated in space so that theircommon secondary structural elements (i.e. alpha-helices andbeta-strands) were oriented such that these common elements were asclose in position to one another as possible. (This superpositioning wasperformed using accepted mathematical formulae rather than actuallyphysically moving the structures by hand.)

Step 3: With the seven structures thus superimposed, for each residue inthe template (2FB4) Fab one calculates the distance from the templatealpha-carbon atom (Cα) to the analogous Cα atom in each of the other sixsuperimposed structures. This results in a table of Cα-Cα distances foreach residue position in the sequence. Such a table is necessary inorder to determine which residue positions will be included in theconsensus model. Generally, if all Cα-Cα distances for a given residueposition were ≦1.0 Å, that position was included in the consensusstructure. If for a given position only one Fab crystal structurewas >1.0 Å, the position was included but the outlying crystal structurewas not included in the next step (for this position only). In general,the seven β-strands were included in the consensus structure while someof the loops connecting the β-strands, e.g. complementarity-determiningregions (CDRs), were not included in view of Cα divergence.

Step 4: For each residue which was included in the consensus structureafter step 3, the average of the coordinates for individual mainchain N,Cα, C, O and Cβ atoms were calculated. Due to the averaging procedure,as well as variation in bond length, bond angle and dihedral angle amongthe crystal structures, this “average” structure contained some bondlengths and angles which deviated from standard geometry. For purposesof this invention, “standard geometry” is understood to includegeometries commonly accepted as typical, such as the compilation of bondlengths and angles from small molecule structures in Weiner, S. J. et.al., J. Amer. Chem. Soc., 106: 765-784 (1984).

Step 5: In order to correct these deviations, the final step was tosubject the “average” structure to 50 cycles of energy minimization(DISCOVER program, Biosym Technologies) using the AMBER (Weiner, S. J.et. al., J. Amer. Chem. Soc., 106: 765-784 (1984)) parameter set withonly the Cα coordinates fixed (i.e. all other atoms are allowed to move)(energy minimization is described below). This allowed any deviant bondlengths and angles to assume a standard (chemically acceptable)geometry. See Table II.

TABLE II Average Bond Lengths and Angles for “Average” (Before) andEnergy-Minimized Consensus (After 50 Cycles) Structures StandardGeometry V_(L)κ before (Å) V_(L)κ after (Å) V_(H) before (Å) V_(H) after(Å) (Å) N—Cα 1.459(0.012) 1.451(0.004) 1.451(0.023) 1.452(0.004) 1.449Cα—C 1.515(0.012) 1.523(0.005) 1.507(0.033) 1.542(0.005) 1.522 O═C1.208(0.062) 1.229(0.003) 1.160(0.177) 1.231(0.003) 1.229 C—N1.288(0.049) 1.337(0.002) 1.282(0.065) 1.335(0.004) 1.335 Cα—Cβ1.508(0.026) 1.530(0.002) 1.499(0.039) 1.530(0.002) 1.526 (*) (*) (*)(*) (*) C—N—Cα 123.5(4.2) 123.8(1.1) 125.3(4.6) 124.0(1.1) 121.9 N—Cα—C110.0(4.0) 109.5(1.9) 110.3(2.8) 109.5(1.6) 110.1 Cα—C—N 116.6(4.0)116.6(1.2) 117.6(5.2) 116.6(0.8) 116.6 O═C—N 123.1(4.1) 123.4(0.6)122.2(4.9) 123.3(0.4) 122.9 N—Cα—Cβ 110.3(2.1) 109.8(0.7) 110.6(2.5)109.8(0.6) 109.5 Cβ—Cα—C 111.4(2.4) 111.1(0.7) 111.2(2.2) 111.1(0.6)111.1 Values in parentheses are standard deviations. Note that whilesome bond length and angle averages did not change appreciably afterenergy-minimization, the corresponding standard deviations are reduceddue to deviant geometries assuming standard values afterenergy-minimization. Standard geometry values are from the AMBERforcefield as implemented in DISCOVER (Biosym Technologies).

The consensus structure might conceivably be dependent upon whichcrystal structure was chosen as the template on which the others weresuperimposed. As a test, the entire procedure was repeated using thecrystal structure with the worst superposition versus 2FB4, i.e. the2HFL Fab structure, as the new template (reference). The two consensusstructures compare favorably (root-mean-squared deviation of 0.11 Å forall N, Cα and C atoms).

Note that the consensus structure only includes mainchain (N, Cα, C, O,Cβ atoms) coordinates for only those residues which are part of aconformation common to all seven X-ray crystal structures. For the Fabstructures, these include the common β-strands (which comprise twoβ-sheets) and a few non-CDR loops which connect these β-strands. Theconsensus structure does not include CDRs or sidechains, both of whichvary in their conformation among the seven structures. Also, note thatthe consensus structure includes only the VL and VH domains.

This consensus structure is used as the archetype. It is not particularto any species, and has only the basic shape without side chains.Starting with this consensus structure the model of any import, human,or humanized Fab can be constructed as follows. Using the amino acidsequence of the particular antibody VL and VH domains of interest, acomputer graphics program (such as INSIGHT, Biosym Technologies) is usedto add sidechains and CDRs to the consensus structure. When a sidechainis added, its conformation is chosen on the basis of known Fab crystalstructures (see the Background section for publications of such crystalstructures) and rotamer libraries (Ponder, J. W. & Richards, F. M., J.Mol. Biol. 193: 775-791 (1987)). The model also is constructed so thatthe atoms of the sidechain are positioned so as to not collide withother atoms in the Fab.

CDRs are added to the model (now having the backbone plus side chains)as follows.

The size (i.e. number of amino acids) of each import CDR is compared tocanonical CDR structures tabulated by Chothia et al., Nature,342:877-883 (1989)) and which were derived from Fab crystals. Each CDRsequence is also reviewed for the presence or absence of certainspecific amino acid residues which are identified by Chothia asstructurally important: e.g. light chain residues 29 (CDR1) and 95(CDR3), and heavy chain residues 26, 27, 29 (CDR1) and 55 (CDR2). Forlight chain CDR2, and heavy chain CDR3, only the size of the CDR iscompared to the Chothia canonical structure. If the size and sequence(i.e. inclusion of the specific, structurally important residues asdenoted by Chothia et al.) of the import CDR agrees in size and has thesame structurally important residues as those of a canonical CDR, thenthe mainchain conformation of the import CDR in the model is taken to bethe same as that of the canonical CDR. This means that the importsequence is assigned the structural configuration of the canonical CDR,which is then incorporated in the evolving model.

However, if no matching canonical CDR can be assigned for the importCDR, then one of two options can be exercised. First, using a programsuch as INSIGHT (Biosym Technologies), the Brookhaven Protein Data Bankcan be searched for loops with a similar size to that of the import CDRand these loops can be evaluated as possible conformations for theimport CDR in the model. Minimally, such loops must exhibit aconformation in which no loop atom overlaps with other protein atoms.Second, one can use available programs which calculate possible loopconformations, assuming a given loop size, using methods such asdescribed by Bruccoleri et al, Nature 335: 564-568 (1988).

When all CDRs and sidechains have been added to the consensus structureto give the final model (import, human or humanized), the model ispreferably subjected to energy minimization using programs which areavailable commercially (e.g. DISCOVER, Biosym Technologies). Thistechnique uses complex mathematical formulae to refine the model byperforming such tasks as checking that all atoms are within appropriatedistances from one another and checking that bond lengths and angles arewithin chemically acceptable limits.

Models of a humanized, import or human antibody sequence are used in thepractice of this invention to understand the impact of selected aminoacid residues of the activity of the sequence being modeled. Forexample, such a model can show residues which may be important inantigen binding, or for maintaining the conformation of the antibody, asdiscussed in more detail below. Modeling can also be used to explore thepotential impact of changing any amino acid residue in the antibodysequence.

Methods for Obtaining a Humanized Antibody Sequence

In the practice of this invention, the first step in humanizing animport antibody is deriving a consensus amino acid sequence into whichto incorporate the import sequences.

Next a model is generated for these sequences using the methodsdescribed above. In certain embodiments of this invention, the consensushuman sequences are derived from the most abundant subclasses in thesequence compilation of Kabat et al. (Kabat, E. A. et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)), namely V_(L) κ subgroup I and V_(H) group III.and have the sequences indicated in the definitions above.

While these steps may be taken in different order, typically a structurefor the candidate humanized antibody is created by transferring the atleast one CDR from the non-human, import sequence into the consensushuman structure, after the entire corresponding human CDR has beenremoved. The humanized antibody may contain human replacements of thenon-human import residues at positions within CDRs as defined bysequence variability (Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987)) or as defined by structural variability (Chothia, C. & Lesk, A.M., J. Mol. Biol. 196:901-917 (1987)). For example, huMAb4D5 containshuman replacements of the muMAb4D5 residues at three positions withinCDRs as defined by sequence variability (Kabat, E. A. et al., Sequencesof Proteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)) but not as defined by structural variability(Chothia, C. & Lesk, A. M., J. Mol. Biol. 196:901-917 (1987)):V_(L)-CDR1 K24R, V_(L)-CDR2 R54L and V_(L)-CDR2 T56S.

Differences between the non-human import and the human consensusframework residues are individually investigated to determine theirpossible influence on CDR conformation and/or binding to antigen.Investigation of such possible influences is desirably performed throughmodeling, by examination of the characteristics of the amino acids atparticular locations, or determined experimentally through evaluatingthe effects of substitution or mutagenesis of particular amino acids.

In certain preferred embodiments of this invention, a humanized antibodyis made comprising amino acid sequence of an import, non-human antibodyand a human antibody, utilizing the steps of:

a. obtaining the amino acid sequences of at least a portion of an importantibody variable domain and of a consensus human variable domain;

b. identifying Complementarity Determining Region (CDR) amino acidsequences in the import and the human variable domain sequences;

c. substituting an import CDR amino acid sequence for the correspondinghuman CDR amino acid sequence;

d. aligning the amino acid sequences of a Framework Region (FR) of theimport antibody and the corresponding FR of the consensus antibody;

e. identifying import antibody FR residues in the aligned FR sequencesthat are non-homologous to the corresponding consensus antibodyresidues;

f. determining if the non-homologous import amino acid residue isreasonably expected to have at least one of the following effects:

1. non-covalently binds antigen directly,

2. interacts with a CDR; or

3. participates in the V_(L)-V_(H) interface; and

g. for any non-homologous import antibody amino acid residue which isreasonably expected to have at least one of these effects, substitutingthat residue for the corresponding amino acid residue in the consensusantibody FR sequence.

Optionally, one determines if any non-homologous residues identified instep (e) are exposed on the surface of the domain or buried within it,and if the residue is exposed but has none of the effects identified instep (f), one may retain the consensus residue.

Additionally, in certain embodiments the corresponding consensusantibody residues identified in step (e) above are selected from thegroup consisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L,64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H,36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H,75H, 76H, 78H, 91H, 92H, 93H, and 103H (utilizing the numbering systemset forth in Kabat, E. A. et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987)).

In preferred embodiments, the method of this invention comprises theadditional steps of searching either or both of the import, non-humanand the consensus variable domain sequences for glycosylation sites,determining if the glycosylation is reasonably expected to be importantfor the desired antigen binding and biological activity of the antibody(i.e., determining if the glycosylation site binds to antigen or changesa side chain of an amino acid residue that binds to antigen, or if theglycosylation enhances or weakens antigen binding, or is important formaintaining antibody affinity). If the import sequence bears theglycosylation site, it is preferred to substitute that site for thecorresponding residues in the consensus human sequence if theglycosylation site is reasonably expected to be important. If only theconsensus sequence, and not the import, bears the glycosylation site, itis preferred to eliminate that glycosylation site or substitute thereforthe corresponding amino acid residues from the import sequence.

Another preferred embodiment of the methods of this invention comprisesaligning import antibody and the consensus antibody FR sequences,identifying import antibody FR residues which are non-homologous withthe aligned consensus FR sequence, and for each such non-homologousimport antibody FR residue, determining if the corresponding consensusantibody residue represents a residue which is highly conserved acrossall species at that site, and if it is so conserved, preparing ahumanized antibody which comprises the consensus antibody amino acidresidue at that site.

In certain alternate embodiments, one need not utilize the modeling andevaluation steps described above, and may instead proceed with the stepsof obtaining the amino acid sequence of at least a portion of an import,non-human antibody variable domain having a CDR and a FR, obtaining theamino acid sequence of at least a portion of a consensus human antibodyvariable domain having a CDR and a FR, substituting the non-human CDRfor the human CDR in the consensus human antibody variable domain, andthen substituting an amino acid residue for the consensus amino acidresidue at at least one of the following sites:

a. (in the FR of the variable domain of the light chain) 4L, 35L, 36L,38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L,71L, 73L, 85L, 87L, 98L, or

b. (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H,36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H,75H, 76H, 78H, 91H, 92H, 93H, and 103H.

Preferably, the non-CDR residue substituted at the consensus FR site isthe residue found at the corresponding location of the non-humanantibody. If desired, one may utilize the other method steps describedabove for determining whether a particular amino acid residue canreasonably be expected to have undesirable effects, and remedying thoseeffects.

If after making a humanized antibody according to the steps above andtesting its activity one is not satisfied with the humanized antibody,one preferably reexamines the potential effects of the amino acids atthe specific locations recited above. Additionally, it is desirable toreinvestigate any buried residues which are reasonably expected toaffect the V_(L)-V_(H) interface but may not directly affect CDRconformation. It is also desirable to reevaluate the humanized antibodyutilizing the steps of the methods claimed herein.

In certain embodiments of this invention, amino acid residues in theconsensus human sequence are substituted for by other amino acidresidues. In preferred embodiments, residues from a particular non-humanimport sequence are substituted, however there are circumstances whereit is desired to evaluate the effects of other amino acids. For example,if after making a humanized antibody according to the steps above andtesting its activity one is not satisfied with the humanized antibody,one may compare the sequences of other classes or subgroups of humanantibodies, or classes or subgroups of antibodies from the particularnon-human species, and determine which other amino acid side chains andamino acid residues are found at particular locations and substitutingsuch other residues.

Antibodies

Certain aspects of this invention are directed to natural antibodies andto monoclonal antibodies, as illustrated in the Examples below and byantibody hybridomas deposited with the ATCC (as described below). Thus,the references throughout this description to the use of monoclonalantibodies are intended to include the use of natural or nativeantibodies as well as humanized and chimeric antibodies. As used herein,the term “antibody” includes the antibody variable domain and otherseparable antibody domains unless specifically excluded.

In accordance with certain aspects of this invention, antibodies to behumanized (import antibodies) are isolated from continuous hybrid celllines formed by the fusion of antigen-primed immune lymphocytes withmyeloma cells.

In certain embodiments, the antibodies of this invention are obtained byroutine screening. Polyclonal antibodies to an antigen generally areraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the antigen and an adjuvant. It may be useful to conjugatethe antigen or a fragment containing the target amino acid sequence to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

The route and schedule of the host animal or cultured antibody-producingcells therefrom are generally in keeping with established andconventional techniques for antibody stimulation and production. Whilemice are frequently employed as the test model, it is contemplated thatany mammalian subject including human subjects or antibody-producingcells obtained therefrom can be manipulated according to the processesof this invention to serve as the basis for production of mammalian,including human, hybrid cell lines.

Animals are typically immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreund's complete adjuvant (or other suitable adjuvant) by subcutaneousinjection at multiple sites 7 to 14 days later animals are bled and theserum is assayed for antigen titer. Animals are boosted until the titerplateaus. Preferably, the animal is boosted with the conjugate of thesame antigen, but conjugated to a different protein and/or through adifferent cross-linking agent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

After immunization, monoclonal antibodies are prepared by recoveringimmune lymphoid cells-typically spleen cells or lymphocytes from lymphnode tissue-from immunized animals and immortalizing the cells inconventional fashion, e.g. by fusion with myeloma cells or byEpstein-Barr (EB)-virus transformation and screening for clonesexpressing the desired antibody.

The hybridoma technique described originally by Kohler and Milstein,Eur. J. Immunol. 6:511 (1976) has been widely applied to produce hybridcell lines that secrete high levels of monoclonal antibodies againstmany specific antigens.

It is possible to fuse cells of one species with another. However, it ispreferable that the source of the immunized antibody producing cells andthe myeloma be from the same species.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody. The secreted antibody is recovered from tissueculture supernatant by conventional methods such as precipitation, Ionexchange chromatography, affinity chromatography, or the like. Theantibodies described herein are also recovered from hybridoma cellcultures by conventional methods for purification of IgG or IgM as thecase may be that heretofore have been used to purify theseimmunoglobulins from pooled plasma, e.g. ethanol or polyethylene glycolprecipitation procedures. The purified antibodies are sterile filtered,and optionally are conjugated to a detectable marker such as an enzymeor spin label for use in diagnostic assays of the antigen in testsamples.

While routinely rodent monoclonal antibodies are used as the source ofthe import antibody, the invention is not limited to any species.Additionally, techniques developed for the production of chimericantibodies (Morrison et al., Proc. Natl. Acad. Sci., 81:6851 (1984);Neuberger et al., Nature 312:604 (1984); Takeda et al., Nature 314:452(1985)) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity (such as ability toactivate human complement and mediate ADCC) can be used; such antibodiesare within the scope of this invention.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules (known as Fab fragments) which bypass thegeneration of monoclonal antibodies are encompassed within the practiceof this invention. One extracts antibody-specific messenger RNAmolecules from immune system cells taken from an immunized animal,transcribes these into complementary DNA (cDNA), and clones the cDNAinto a bacterial expressions system. One example of such a techniquesuitable for the practice of this invention was developed by researchersat Scripps/Stratagene, and incorporates a proprietary bacteriophagelambda vector system which contains a leader sequence that causes theexpressed Fab protein to migrate to the periplasmic space (between thebacterial cell membrane and the cell wall) or to be secreted. One canrapidly generate and screen great numbers of functional FAb fragmentsfor those which bind the antigen. Such FAb fragments with specificityfor the antigen are specifically encompassed within the term “antibody”as it is defined, discussed, and claimed herein.

Amino Acid Seauence Variants

Amino acid sequence variants of the antibodies and polypeptides of thisinvention (referred to in herein as the target polypeptide) are preparedby introducing appropriate nucleotide changes into the DNA encoding thetarget polypeptide, or by in vitro synthesis of the desired targetpolypeptide. Such variants include, for example, humanized variants ofnon-human antibodies, as well as deletions from, or insertions orsubstitutions of, residues within particular amino acid sequences. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes also mayalter post-translational processes of the target polypeptide, such aschanging the number or position of glycosylation sites, altering anymembrane anchoring characteristics, and/or altering the intra-cellularlocation of the target polypeptide by inserting, deleting, or otherwiseaffecting any leader sequence of the native target polypeptide.

In designing amino acid sequence variants of target polypeptides, thelocation of the mutation site and the nature of the mutation will dependon the target polypeptide characteristic(s) to be modified. The sitesfor mutation can be modified individually or in series, e.g., by (1)substituting first with conservative amino acid choices and then withmore radical selections depending upon the results achieved, (2)deleting the target residue, or (3) inserting residues of the same or adifferent class adjacent to the located site, or combinations of options1-3. In certain embodiments, these choices are guided by the methods forcreating humanized sequences set forth above.

A useful method for identification of certain residues or regions of thetarget polypeptide that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells (Science, 244: 1081-1085 [1989]). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis may be conducted at the target codon or region andthe expressed target polypeptide variants are screened for the optimalcombination of desired activity.

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. In general, the location and nature of the mutation chosenwill depend upon the target polypeptide characteristic to be modified.

Amino acid sequence deletions of antibodies are generally not preferred,as maintaining the generally configuration of an antibody is believed tobe necessary for its activity. Any deletions will be selected so as topreserve the structure of the target antibody.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the target polypeptide sequence) may range generallyfrom about 1 to 10 residues, more preferably 1 to 5, most preferably 1to 3. Examples of terminal insertions include the target polypeptidewith an N-terminal methionyl residue, an artifact of the directexpression of target polypeptide in bacterial recombinant cell culture,and fusion of a heterologous N-terminal signal sequence to theN-terminus of the target polypeptide molecule to facilitate thesecretion of the mature target polypeptide from recombinant host cells.Such signal sequences generally will be obtained from, and thushomologous to, the intended host cell species. Suitable sequencesinclude STII or Ipp for E. coli, alpha factor for yeast, and viralsignals such as herpes gD for mammalian cells.

Other insertional variants of the target polypeptide include the fusionto the N- or C-terminus of the target polypeptide of immunogenicpolypeptides, e.g., bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin constant regions (or other immunoglobulin regions),albumin, or ferritin, as described in WO 89102922 published Apr. 6,1989.

Another group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the target polypeptidemolecule removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of the target polypeptide, and siteswhere the amino acids found in the target polypeptide from variousspecies are substantially different in terms of side-chain bulk, charge,and/or hydrophobicity. Other sites for substitution are described infra,considering the effect of the substitution of the antigen binding,affinity and other characteristics of a particular target antibody.

Other sites of interest are those in which particular residues of thetarget polypeptides obtained from various species are identical. Thesepositions may be important for the biological activity of the targetpolypeptide. These sites, especially those falling within a sequence ofat least three other identically conserved sites, are substituted in arelatively conservative manner. If such substitutions result in a changein biological activity, then other changes are introduced and theproducts screened until the desired effect is obtained.

Substantial modifications in function or immunological identity of thetarget polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues may be introducedinto regions of the target polypeptide that are homologous with otherantibodies of the same class or subclass, or, more preferably, into thenon-homologous regions of the molecule.

Any cysteine residues not involved in maintaining the properconformation of target polypeptide also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant crosslinking.

DNA encoding amino acid sequence variants of the target polypeptide isprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants) or preparationby oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the target polypeptide. A particularlypreferred method of gene conversion mutagenesis is described below inExample 1. These techniques may utilized target polypeptide nucleic acid(DNA or RNA), or nucleic acid complementary to the target polypeptidenucleic acid. Oligonucleotide-mediated mutagenesis is a preferred methodfor preparing substitution, deletion, and insertion variants of targetpolypeptide DNA. This technique is well known in the art as described byAdelman et al., DNA, 2: 183 (1983). Briefly, the target polypeptide DNAis altered by hybridizing an oligonucleotide encoding the desiredmutation to a DNA template, where the template is the single-strandedform of a plasmid or bacteriophage containing the unaltered or nativeDNA sequence of the target polypeptide. After hybridization, a DNApolymerase is used to synthesize an entire second complementary strandof the template that will thus incorporate the oligonucleotide primer,and will code for the selected alteration in the target polypeptide DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765 [1978]).

Single-stranded DNA template may also be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of the target polypeptide, and the other strand (the originaltemplate) encodes the native, unaltered sequence of the targetpolypeptide. This heteroduplex molecule is then transformed into asuitable host cell, usually a prokaryote such as E. coli JM 101. Afterthe cells are grown, they are plated onto agarose plates and screenedusing the oligonucleotide primer radiolabeled with 32-phosphate toidentify the bacterial colonies that contain the mutated DNA. Themutated region is then removed and placed in an appropriate vector forprotein production, generally an expression vector of the type typicallyemployed for transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex.

Upon addition of DNA polymerase to this mixture, a strand of DNAidentical to the template except for the mutated bases is generated. Inaddition, this new strand of DNA will contain dCTP-(aS) instead of dCTP,which serves to protect it from restriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding target polypeptide variants with more than one amino acidto be substituted may be generated in one of several ways. If the aminoacids are located close together in the polypeptide chain, they may bemutated simultaneously using one oligonucleotide that codes for all ofthe desired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid variants oftarget polypeptide. While the following discussion refers to DNA, it isunderstood that the technique also finds application with RNA. The PCRtechnique generally refers to the following procedure (see Erlich,supra, the chapter by R. Higuchi, p. 61-70): When small amounts oftemplate DNA are used as starting material in a PCR, primers that differslightly in sequence from the corresponding region in a template DNA canbe used to generate relatively large quantities of a specific DNAfragment that differs from the template sequence only at the positionswhere the primers differ from the template. For introduction of amutation into a plasmid DNA, one of the primers is designed to overlapthe position of the mutation and to contain the mutation; the sequenceof the other primer must be identical to a stretch of sequence of theopposite strand of the plasmid, but this sequence can be locatedanywhere along the plasmid DNA. It is preferred, however, that thesequence of the second primer is located within 200 nucleotides fromthat of the first, such that in the end the entire amplified region ofDNA bounded by the primers can be easily sequenced. PCR amplificationusing a primer pair like the one just described results in a populationof DNA fragments that differ at the position of the mutation specifiedby the primer, and possibly at other positions, as template copying issomewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide tri-phosphates andis included in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayed with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/μl, purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 min.at 55° C., then 30 sec. at 72° C., then 19 cycles of the following: 30sec. at 94° C., 30 sec. at 55° C., and 30 sec. at 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50:vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34: 315 [1985]). Thestarting material is the plasmid (or other vector) comprising the targetpolypeptide DNA to be mutated. The codon(s) in the target polypeptideDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the target polypeptide DNA. After therestriction sites have been introduced into the plasmid, the plasmid iscut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated target polypeptide DNA sequence.

Insertion of DNA into a Cloning Vehicle

The cDNA or genomic DNA encoding the target polypeptide is inserted intoa replicable vector for further cloning (amplification of the DNA) orfor expression. Many vectors are available, and selection of theappropriate vector will depend on 1) whether it is to be used for DNAamplification or for DNA expression, 2) the size of the DNA to beinserted into the vector, and 3) the host cell to be transformed withthe vector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the host cellfor which it is compatible. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(a) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of the target polypeptide DNA that is inserted into thevector.

The target polypeptides of this invention may be expressed not onlydirectly, but also as a fusion with a heterologous polypeptide,preferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. Included within the scope of this invention are targetpolypeptides with any native signal sequence deleted and replaced with aheterologous signal sequence. The heterologous signal sequence selectedshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native target polypeptide signal sequence, thesignal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeastsecretion the native target polypeptide signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

(b) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the target polypeptide DNA. However, the recovery ofgenomic DNA encoding the target polypeptide is more complex than that ofan exogenously replicated vector because restriction enzyme digestion isrequired to excise the target polypeptide DNA.

(c) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, ortetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g. the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327[1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980])or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 [1985]). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thetarget polypeptide nucleic acid, such as dihydrofolate reductase (DHFR)or thymidine kinase. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the target polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the targetpolypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 [1980]. The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingthe target polypeptide.

This amplification technique can be used with any otherwise suitablehost, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060). Alternatively, host cells(particularly wild-type hosts that contain endogenous DHFR) transformedor co-transformed with DNA sequences encoding the target polypeptide,wild-type DHFR protein, and another selectable marker such asaminoglycoside 3′ phosphotransferase (APH) can be selected by cellgrowth in medium containing a selection agent for the selectable markersuch as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, orG418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 [1979];Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al., Gene, 10: 157[1980]). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(d) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the targetpolypeptide nucleic acid. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofa particular nucleic acid sequence, such as that encoding the targetpolypeptide, to which they are operably linked. Such promoters typicallyfall into two classes, inducible and constitutive. Inducible promotersare promoters that initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,e.g. the presence or absence of a nutrient or a change in temperature.At this time a large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto DNA encoding the target polypeptide by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector. Both the native target polypeptidepromoter sequence and many heterologous promoters may be used to directamplification and/or expression of the target polypeptide DNA. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of expressed target polypeptide ascompared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al. Nature, 275: 615[1978]; and Goeddel et al., Nature , 281: 544[1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the target polypeptide(Siebenlist et al., Cell, 20: 269 [1980]) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also generally will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the target polypeptide.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al, J.Adv. Enzyme Reg., 7: 149 [1968]; and Holland,Biochemistry,17:4900[1978]), such asenolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, met allothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

Target polypeptide transcription from vectors in mammalian host cells iscontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with thetarget polypeptide sequence, provided such promoters are compatible withthe host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78: 7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295: 503-508 (1982) on expressing cDNA encoding immuneinterferon in monkey cells; , Reyes et al., Nature, 297: 598-601 (1982)on expression of human β-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus,Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) onexpression of the human interferon β1 gene in cultured mouse and rabbitcells, and Gorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781(1982) on expression of bacterial CAT sequences in CV-1 monkey kidneycells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLacells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

(e) Enhancer Element Component

Transcription of DNA encoding the target polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10-300 bp, that act on a promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent having been found 5′ (Laimins et al., Proc. Natl. Acad. Sci.USA, 78: 993 [1981]) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108[1983]) to the transcription unit, within an intron (Banerji et al.,Cell, 33: 729 [1983]) as well as within the coding sequence itself(Osborne et al., Mol. Cell Bio. 4: 1293 [1984]). Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein and insulin). Typically, however, one will use an enhancerfrom a eukaryotic cell virus. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100-270), the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. See also Yaniv, Nature,297: 17-18 (1982) on enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the target polypeptide DNA, but is preferably located at a site5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNA or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the target polypeptide. The 3′ untranslatedregions also include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components the desired coding and control sequences employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology. 65: 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the target polypeptide. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of the targetpolypeptide that have target polypeptide-like activity.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the target polypeptide in recombinant vertebrate cellculture are described in Gething et al., Nature, 293: 620-625 [1981];Mantei et al., Nature, 281:40-46 [1979]; Levinson et al.; EP 117,060;and EP 117,058. A particularly useful plasmid for mammalian cell cultureexpression of the target polypeptide is pRK5 (EP pub. no. 307,247) orpSVI6B.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescans. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli χ1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.

These examples are illustrative rather than limiting. Preferably thehost cell should secrete minimal amounts of proteolytic enzymes.Alternatively, in vitro methods of cloning, e.g. PCR or other nucleicacid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for target polypeptide-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe [Beach and Nurse,Nature, 290: 140 (1981); EP 139,383 published May 2, 1985],Kluyveromyces hosts (U.S. Pat. No. 4,943,529) such as, e.g., K. lactis[Louvencourt et al., J. Bacteriol., 737 (1983)], K. fragilis, K.bulgaricus, K. thermotolerans, and K. marxianus, yarrowia [EP 402,226],Pichia pastoris [EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265-278 (1988)], Candida, Trichoderma reesia [EP 244,234],Neurospora crassa [Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)], and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium [WO 91/00357 published Jan. 10, 1991], andAspergillus hosts such as A. nidulans [Ballance et al., Biochem.Biophys. Res. Commun. 112: 284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474(1984)] and A. niger [Kelly and Hynes, EMBO J., 4:475479 (1985)].

Suitable host cells for the expression of glycosylated targetpolypeptide are derived from multicellular organisms. Such host cellsare capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori host cells have been identified. See, e.g., Luckow et al.,Bio/Technology, 6: 47-55 (1988); Miller et al., in Genetic Engineering,Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing, 1986), pp.277-279; and Maeda et al., Nature, 315: 592-594 (1985). A variety ofsuch viral strains are publicly available, e.g., the L-1 variant ofAutographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, andsuch viruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the target polypeptide DNA. During incubation of the plant cellculture with A. tumefaciens, the DNA encoding target polypeptide istransferred to the plant cell host such that it is transfected, andwill, under appropriate conditions, express the target polypeptide DNA.In addition, regulatory and signal sequences compatible with plant cellsare available, such as the nopaline synthase promoter andpolyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen.,1: 561 (1982). In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. See EP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years [Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36: 59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251[1980]); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatomacell line (Hep G2). Preferred host cells are human embryonic kidney 293and Chinese hamster ovary cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 publishedJun. 29, 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in sections 16.30-16.37 ofSambrook et al, supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829(1979). However, other methods for introducing DNA into cells such as bynuclear injection, electroporation, or protoplast fusion may also beused.

Culturing the Host Cells

Prokaryotic cells used to produce the target polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the target polypeptide of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium([DMEM], Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz., 58: 44(1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. Re. 30,985, may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

It is further envisioned that the target polypeptides of this inventionmay be produced by homologous recombination, or with recombinantproduction methods utilizing control elements introduced into cellsalready containing DNA encoding the target polypeptide currently in usein the field. For example, a powerful promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredtarget polypeptide. The control element does not encode the targetpolypeptide of this invention, but the DNA is present in the host cellgenome. One next screens for cells making the target polypeptide of thisinvention, or increased or decreased levels of expression, as desired.

Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³²P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native target polypeptide or against a synthetic peptide basedon the DNA sequences provided herein as described further in Section 4below.

Purification of the Target polypeptide

The target polypeptide preferably is recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates when directly expressed without a secretory signal.

When the target polypeptide is expressed in a recombinant cell otherthan one of human origin, the target polypeptide is completely free ofproteins or polypeptides of human origin. However, it is necessary topurify the target polypeptide from recombinant cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to the target polypeptide. As a first step, the culture medium orlysate is centrifuged to remove particulate cell debris. The membraneand soluble protein fractions are then separated. The target polypeptidemay then be purified from the soluble protein fraction and from themembrane fraction of the culture lysate, depending on whether the targetpolypeptide is membrane bound. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG.

Target polypeptide variants in which residues have been deleted,inserted or substituted are recovered in the same fashion, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a target polypeptide fusion withanother protein or polypeptide, e.g. a bacterial or viral antigen,facilitates purification; an immunoaffinity column containing antibodyto the antigen (or containing antigen, where the target polypeptide isan antibody) can be used to adsorb the fusion. Immunoaffinity columnssuch as a rabbit polyclonal anti-target polypeptide column can beemployed to absorb the target polypeptide variant by binding it to atleast one remaining immune epitope. A protease inhibitor such as phenylmethyl sulfonyl fluoride (PMSF) also may be useful to inhibitproteolytic degradation during purification, and antibiotics may beincluded to prevent the growth of adventitious contaminants. One skilledin the art will appreciate that purification methods suitable for nativetarget polypeptide may require modification to account for changes inthe character of the target polypeptide or its variants upon expressionin recombinant cell culture.

Covalent Modifications of Target Polypeptides

Covalent modifications of target polypeptides are included within thescope of this invention. One type of covalent modification includedwithin the scope of this invention is a target polypeptide fragment.Target polypeptide fragments having up to about 40 amino acid residuesmay be conveniently prepared by chemical synthesis, or by enzymatic orchemical cleavage of the full-length target polypeptide or varianttarget polypeptide. Other types of covalent modifications of the targetpolypeptide or fragments thereof are introduced into the molecule byreacting specific amino acid residues of the target polypeptide orfragments thereof with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride: trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingtarget polypeptide to a water-insoluble support matrix or surface foruse in the method for purifying anti-target polypeptide antibodies, andvice versa. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the target polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. By altering is meant deletingone or more carbohydrate moieties found in the native targetpolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native target polypeptide.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the target polypeptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the native target polypeptide sequence (for O-linkedglycosylation sites). For ease, the target polypeptide amino acidsequence is preferably altered through changes at the DNA level,particularly by mutating the DNA encoding the target polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above under the heading of “Amino Acid SequenceVariants of Target Polypeptide”.

Another means of increasing the number of carbohydrate moieties on thetarget polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. These procedures are advantageous in that they do notrequire production of the polypeptide in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem., pp. 259-306[1981]).

Removal of carbohydrate moieties present on the native targetpolypeptide may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al. (Arch. Biochem. Biophys., 259:52 [1987]) and by Edge et al.(Anal. Biochem., 118:131 [1981]). Enzymatic cleavage of carbohydratemoieties on polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al. (Meth.Enzymol., 138:350 [1987]). Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al. (J. Biol. Chem., 257:3105 [1982]).Tunicamycin blocks the formation of protein-N-glycoside linkages.

Another type of covalent modification of the target polypeptidecomprises linking the target polypeptide to various nonproteinaceouspolymers, e.g. polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The target polypeptide also may be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Reminaton's Pharmaceutical Sciences,16th edition, Osol, A., Ed., (1980).

Target polypeptide preparations are also useful in generatingantibodies, for screening for binding partners, as standards in assaysfor the target polypeptide (e.g. by labeling the target polypeptide foruse as a standard in a radioimmunoassay, enzyme-linked immunoassay, orradioreceptor assay), in affinity purification techniques, and incompetitive-type receptor binding assays when labeled with radioiodine,enzymes, fluorophores, spin labels, and the like.

Since it is often difficult to predict in advance the characteristics ofa variant target polypeptide, it will be appreciated that some screeningof the recovered variant will be needed to select the optimal variant.For example, a change in the immunological character of the targetpolypeptide molecule, such as affinity for a given antigen or antibody,is measured by a competitive-type immunoassay. The variant is assayedfor changes in the suppression or enhancement of its activity bycomparison to the activity observed for the target polypeptide s in thesame assay. Other potential modifications of protein or polypeptideproperties such as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, stability in recombinant cellculture or in plasma, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

Diagnostic and Related Uses of the Antibodies

The antibodies of this invention are useful in diagnostic assays forantigen expression in specific cells or tissues. The antibodies aredetectably labeled and/or are immobilized on an insoluble matrix.

The antibodies of this invention find further use for the affinitypurification of the antigen from recombinant cell culture or naturalsources. Suitable diagnostic assays for the antigen and its antibodiesdepend on the particular antigen or antibody. Generally, such assaysinclude competitive and sandwich assays, and steric inhibition assays.Competitive and sandwich methods employ a phase-separation step as anintegral part of the method while steric inhibition assays are conductedin a single reaction mixture. Fundamentally, the same procedures areused for the assay of the antigen and for substances that bind theantigen, although certain methods will be favored depending upon themolecular weight of the substance being assayed. Therefore, thesubstance to be tested is referred to herein as an analyte, irrespectiveof its status otherwise as an antigen or antibody, and proteins thatbind to the analyte are denominated binding partners, whether they beantibodies, cell surface receptors, or antigens.

Analytical methods for the antigen or its antibodies all use one or moreof the following reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner andsteric conjugates. The labeled reagents also are known as “tracers.”

The label used (and this is also useful to label antigen nucleic acidfor use as a probe) is any detectable functionality that does notinterfere with the binding of analyte and its binding partner. Numerouslabels are known for use in immunoassay, examples including moietiesthat may be detected directly, such as fluorochrome, chemiluminescent,and radioactive labels, as well as moieties, such as enzymes, that mustbe reacted or derivatized to be detected. Examples of such labelsinclude the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophoressuch as rare earth chelates or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, umbelliferone, luceriferases,e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradishperoxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase,lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase,and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such asuricase and xanthine oxidase, coupled with an enzyme that employshydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. Forinstance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al., “Methodsfor the Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.Such bonding methods are suitable for use with the antibodies andpolypeptides of this invention.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, the antigen or its immunologically active fragments areconjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with antibody so thatbinding of the antibody inhibits or potentiates the enzyme activity ofthe label. This method per se is widely practiced under the name ofEMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of antigenor antibodies. In sequential sandwich assays an immobilized bindingpartner is used to adsorb test sample analyte, the test sample isremoved as by washing, the bound analyte is used to adsorb labeledbinding partner, and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample analyte. In “simultaneous” sandwich assays the test sample is notseparated before adding the labeled binding partner. A sequentialsandwich assay using an anti-antigen monoclonal antibody as one antibodyand a polyclonal anti-antigen antibody as the other is useful in testingsamples for particular antigen activity.

The foregoing are merely exemplary diagnostic assays for the import andhumanized antibodies of this invention. Other methods now or hereafterdeveloped for the determination of these analytes are included withinthe scope hereof, including the bioassays described above.

Immunotoxins

This invention is also directed to immunochemical derivatives of theantibodies of this invention such as immunotoxins (conjugates of theantibody and a cytotoxic moiety).

Antibodies which carry the appropriate effector functions, such as withtheir constant domains, are also used to induce lysis through thenatural complement process, and to interact with antibody dependentcytotoxic cells normally present.

For example, purified, sterile filtered antibodies are optionallyconjugated to a cytotoxin such as ricin for use in AIDS therapy. U.S.patent application Ser. No. 07/350,895 illustrates methods for makingand using immunotoxins for the treatment of HIV infection. The methodsof this invention, for example, are suitable for obtaining humanizedantibodies for use as immunotoxins for use in AIDS therapy.

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or anenzymatically active toxin of bacterial, fungal, plant or animal origin,or an enzymatically active fragment of such a toxin. Enzymaticallyactive toxins and fragments thereof used are diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Inanother embodiment, the antibodies are conjugated to small moleculeanticancer drugs such as cis-platin or 5FU.

Conjugates of the monoclonal antibody and such cytotoxic moieties aremade using a variety of bifunctional protein coupling agents. Examplesof such reagents are SPDP, IT, bifunctional derivatives of imidoesterssuch as dimethyl adipimidate HCl, active esters such as disuccinimidylsuberate, aldehydes such as glutaraldehyde, bis-azido compounds such asbis (p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such asbis-(p-diazoniumbenzoyl)--ethylenediamine, diisocyanates such astolylene 2,6-diisocyanate and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may bejoined to the Fab fragment of the antibodies.

Immunotoxins can be made in a variety of ways, as discussed herein.Commonly known crosslinking reagents can be used to yield stableconjugates.

Advantageously, monoclonal antibodies specifically binding the domain ofthe antigen which is exposed on the infected cell surface, areconjugated to ricin A chain. Most advantageously the ricin A chain isdeglycosylated and produced through recombinant means. All advantageousmethod of making the ricin immunotoxin is described in Vitetta et al.,Science 238:1098 (1987).

When used to kill infected human cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques.

Cytotoxic radiopharmaceuticals for treating infected cells may be madeby conjugating radioactive isotopes (e.g. I, Y, Pr) to the antibodies.Advantageously alpha particle-emitting isotopes are used. The term“cytotoxic moiety” as used herein is intended to include such isotopes.

In a preferred embodiment, ricin A chain is deglycosylated or producedwithout oligosaccharides, to decrease its clearance by irrelevantclearance mechanisms (e.g., the liver). In another embodiment, wholericin (A chain plus B chain) is conjugated to antibody if the galactosebinding property of B-chain can be blocked (“blocked ricin”).

In a further embodiment toxin-conjugates are made with Fab or F(ab′)₂fragments.

Because of their relatively small size these fragments can betterpenetrate tissue to reach infected cells.

In another embodiment, fusogenic liposomes are filled with a cytotoxicdrug and the liposomes are coated with antibodies specifically bindingthe particular antigen.

Antibody Dependent Cellular Cytotoxicity

Certain aspects of this invention involve antibodies which are (a)directed against a particular antigen and (b) belong to a subclass orisotype that is capable of mediating the lysis of cells to which theantibody molecule binds. More specifically, these antibodies shouldbelong to a subclass or isotype that, upon complexing with cell surfaceproteins, activates serum complement and/or mediates antibody dependentcellular cytotoxicity (ADCC) by activating effector cells such asnatural killer cells or macrophages.

Biological activity of antibodies is known to be determined, to a largeextent, by the constant domains or Fc region of the antibody molecule(Uananue and Benacerraf, Textbook of Immunology, 2nd Edition, Williams &Wilkins, p. 218 (1984)). This includes their ability to activatecomplement and to mediate antibody-dependent cellular cytotoxicity(ADCC) as effected by leukocytes. Antibodies of different classes andsubclasses differ in this respect, as do antibodies from the samesubclass but different species; according to the present invention,antibodies of those classes having the desired biological activity areprepared.

Preparation of these antibodies involves the selection of antibodyconstant domains are their incorporation in the humanized antibody byknown technique. For example, mouse immunoglobulins of the IgG3 andIgG2a class are capable of activating serum complement upon binding tothe target cells which express the cognate antigen, and thereforehumanized antibodies which incorporate IgG3 and IgG2a effector functionsare desirable for certain therapeutic applications.

In general, mouse antibodies of the IgG2a and IgG3 subclass andoccasionally IgG1 can mediate ADCC, and antibodies of the IgG3, IgG2a,and IgM subclasses bind and activate serum complement. Complementactivation generally requires the binding of at least two IgG moleculesin closeproximity on the target cell. However, the binding of only oneIgM molecule activates serum complement.

The ability of any particular antibody to mediate lysis of the targetcell by complement activation and/or ADCC can be assayed. The cells ofinterest are grown and labeled in vitro; the antibody is added to thecell culture in combination with either serum complement or immune cellswhich may be activated by the antigen antibody complexes. Cytolysis ofthe target cells is detected by the release of label from the lysedcells. In fact, antibodies can be screened using the patient's own serumas a source of complement and/or immune cells. The antibody that iscapable of activating complement or mediating ADCC in the in vitro testcan then be used therapeutically in that particular patient.

This invention specifically encompasses consensus Fc antibody domainsprepared and used according to the teachings of this invention.

Therapeutic and Other Uses of the Antibodies

When used in vivo for therapy, the antibodies of the subject inventionare administered to the patient in therapeutically effective amounts(i.e. amounts that have desired therapeutic effect). They will normallybe administered parenterally. The dose and dosage regimen will dependupon the degree of the infection, the characteristics of the particularantibody or immunotoxin used, e.g., its therapeutic index, the patient,and the patient's history. Advantageously the antibody or immunotoxin isadministered continuously over a period of 1-2 weeks, intravenously totreat cells in the vasculature and subcutaneously and intraperitoneallyto treat regional lymph nodes. Optionally, the administration is madeduring the course of adjunct therapy such as combined cycles ofradiation, chemotherapeutic treatment, or administration of tumornecrosis factor, interferon or other cytoprotective or immunomodulatoryagent.

For parenteral administration the antibodies will be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic, and non-therapeutic. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate can also be used. Liposomes may be used as carriers. The vehiclemay contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies will typically be formulated in such vehicles atconcentrations of about 1 mg/ml to 10 mg/ml.

Use of IgM antibodies may be preferred for certain applications, howeverIgG molecules by being smaller may be more able than IgM molecules tolocalize to certain types of infected cells.

There is evidence that complement activation in vivo leads to a varietyof biological effects, including the induction of an inflammatoryresponse and the activation of macrophages (Uananue and Benecerraf,Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).The increased vasodilation accompanying inflammation may increase theability of various agents to localize in infected cells. Therefore,antigen-antibody combinations of the type specified by this inventioncan be used therapeutically in many ways. Additionally, purifiedantigens (Hakomori, Ann. Rev. Immunol. 2:103 (1984)) or anti-idiotypicantibodies (Nepom et al., Proc. Natl. Acad. Sci. 81:2864 (1985);Koprowski et al., Proc. Natl. Acad. Sci. 81:216 (1984)) relating to suchantigens could be used to induce an active immune response in humanpatients. Such a response includes the formation of antibodies capableof activating human complement and mediating ADCC and by such mechanismscause infected cell destruction.

Optionally, the antibodies of this invention are useful in passivelyimmunizing patients, as exemplified by the administration of humanizedanti-HIV antibodies.

The antibody compositions used in therapy are formulated and dosagesestablished in a fashion consistent with good medical practice takinginto account the disorder to be treated, the condition of the individualpatient, the site of delivery of the composition, the method ofadministration and other factors known to practitioners. The antibodycompositions are prepared for administration according to thedescription of preparation of polypeptides for administration, infra.

Deposit of Materials

As described above, cultures of the muMAb4D5 have been deposited withthe American Type Culture Collection 10801 University Blvd., Manassas,Va., USA (ATCC).

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of the deposit. The organisms will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC § 122 and the Commissioner's rules pursuant thereto (including37 CFR §1.12 with particular reference to 886 OG 638).

In respect of those designations in which a European patent is sought, asample of the deposited microorganism will be made available until thepublication of the mention of the grant of the European patent or untilthe date on which the application has been refused or withdrawn or isdeemed to be withdrawn, only by the issue of such a sample to an expertnominated by the person requesting the sample. (Rule 28(4) EPC).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiments are intended to illustrate only certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture appear below, but should not be construed to limit theinvention.

EXAMPLES Example 1 Humanization of muMAb4D5

Here we report the chimerization of muMAb4D5 (chMAb4D5) and the rapidand simultaneous humanization of heavy (V_(H)) and light (V_(L)) chainvariable region genes using a novel “gene conversion mutagenesis”strategy. Eight humanized variants (huMAb4D5) were constructed to probethe importance of several FR residues identified by our molecularmodeling or previously proposed to be critical to the conformation ofparticular CDRs (see Chothia, C. & Lesk, A. M., J. Mol. Biol.196:901-917 (1987); Chothia, C. et al., Nature 342:877-883 (1989);Tramontano, A. et al., J. Mol. Biol. 215:175-182 (1990)). Efficienttransient expression of humanized variants in non-myeloma cells allowedus to rapidly investigate the relationship between binding affinity forp185^(HER2) ECD and anti-proliferative activity against p185^(HER2)overexpressing carcinoma cells.

Materials and Methods

Cloning of Variable Region Genes. The muMAb4D5 V_(H) and V_(L) geneswere isolated by polymerase chain reaction (PCR) amplification of mRNAfrom the corresponding hybridoma (Fendly, B. M. et al., Cancer Res.50:1550-1558 (1990)) as described by Orlandi et al. (Orlandi, R. et al.,Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989)). Amino terminalsequencing of muMAb4D5 V_(L) and V_(H) was used to design the sensestrand PCR primers, whereas the anti-sense PCR primers were based uponconsensus sequences of murine framework residues (Orlandi, R. et al.,Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989); Kabat, E. A. et al.,Sequences of Proteins of Immifnological Interest (National Institutes ofHealth, Bethesda, Md., 1987)) incorporating restriction sites fordirectional cloning shown by underlining and listed after the sequences:V_(L) sense, 5′-TCCGATATCCAGCTGACCCAGTCTCCA-3′ (SEQ ID NO. 7), EcoRV;V_(L) anti-sense, 5′-GTTTGATCTCCAGCTTGGTACCHSCDCCGAA-3′ (SEQ. ID NO. 8),Asp7l8; V_(H) sense, 5′-AGGTSMARCTGCAGSAGTCWGG-3′ (SEQ. ID NO. 9), PstIand V_(H) anti-sense, 5′-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3′ (SEQ. IDNO. 10), BstEII; where H=A or C or T, S=C or G, D=A or G or T, M=A or C,R=A or G and W=A or T. The PCR products were cloned into pUC119 (Vieira,J. & Messing, J., Methods Enzymol. 153:3-11 (1987)) and five clones foreach variable domain sequenced by the dideoxy method (Sanger, F. et al.,Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)).

Molecular Modelling. Models for muMAb4D5 V_(H) and V_(L) domains wereconstructed separately from consensus coordinates based upon seven Fabstructures from the Brookhaven protein data bank (entries 1FB4, 2RHE,2MCP, 3FAB, 1FBJ, 2HFL and 1REI). The Fab fragment KOL (Marquart, M. etal., J. Mol. Biol. 141:369-391 (1980)) was first chosen as a templatefor V_(L) and V_(H) domains and additional structures were thensuperimposed upon this structure using their main chain atom coordinates(INSIGHT program, Biosym Technologies).

The distance from the template Cα to the analogous Cα in each of thesuperimposed structures was calculated for each residue position. If all(or nearly all) Cα-Cα distances for a given residue were ≦1 Å, then thatposition was included in the consensus structure. In most cases theβ-sheet framework residues satisfied these criteria whereas the CDRloops did not. For each of these selected residues the averagecoordinates for individual N, Cα, C, O and Cβ atoms were calculated andthen corrected for resultant deviations from non-standard bond geometryby 50 cycles of energy minimization using the DISCOVER program (BiosymTechnologies) with the AMBER forcefield (Weiner, S. J. et al., J. Amer.Chem. Soc. 106:765-784 (1984)) and Cα coordinates fixed. The side chainsof highly conserved residues, such as the disulfide-bridged cysteineresidues, were then incorporated into the resultant consensus structure.Next the sequences of muMAb4D5 V_(L) and V_(H) were incorporatedstarting with the CDR residues and using the tabulations of CDRconformations from Chothia et al. (Chothia, C. et al., Nature342:877-883 (1989)) as a guide. Side-chain conformations were chosen onthe basis of Fab crystal structures, rotamer libraries (Ponder, J. W. &Richards, F. M., J. Mol. Biol. 193:775-791 (1987)) and packingconsiderations. Since V_(H)-CDR3 could not be assigned a definitebackbone conformation from these criteria, two models were created froma search of similar sized loops using the INSIGHT program. A third modelwas derived using packing and solvent exposure considerations. Eachmodel was then subjected to 5000 cycles of energy minimization.

In humanizing muMAb4D5, consensus human sequences were first derivedfrom the most abundant subclasses in the sequence compilation of Kabatet al. (Kabat, E. A. et al., Sequences of Proteins of immunologicalInterest (National Institutes of Health, Bethesda, Md., 1987)), namelyV_(L) κ subgroup I and V_(H) group III, and a molecular model generatedfor these sequences using the methods described above. A structure forhuMAb4D5 was created by transferring the CDRs from the muMAb4D5 modelinto the consensus human structure. All huMAb4D5 variants contain humanreplacements of muMAb4D5 residues at three positions within CDRs asdefined by sequence variability (Kabat, E. A. et al., Sequences ofProteins of Immunological interest (National Institutes of Health,Bethesda, Md., 1987)) but not as defined by structural variability(Chothia, C. & Lesk, A. M., J. Mol. Biol. 196:901-917 (1987)):V_(L)-CDR1 K24R, V_(L)-CDR2 R54L and V_(L)-CDR2 T56S. Differencesbetween muMAb4D5 and the human consensus framework residues (FIG. 1)were individually modeled to investigate their possible influence on CDRconformation and/or binding to the p185^(HER2) ECD.

Construction of Chimeric Genes. Genes encoding chMAb4D5 light and heavychains were separately assembled in previously described phagemidvectors containing the human cytomegalovirus enhancer and promoter, a 5′intron and SV40 polyadenylation signal (Gorman, C. M. et al., DNA &Prot. Engin. Tech. 2:3-10 (1990)). Briefly, gene segments encodingmuMAb4D5 V_(L) (FIG. 1A) and REI human κ₁ light chain CL (Palm, W. &Hilschmann, N., Z. Physiol. Chem. 356:167-191 (1975)) were preciselyjoined as were genes for muMAb4D5 V_(H) (FIG. 1B) and human γ1 constantregion (Capon, D. J. et al., Nature 337:525-531 (1989)) by simplesubcloning (Boyle, A., in Current Protocols in Molecular Biology,Chapter 3 (F. A. Ausubel et al., eds., Greene Publishing &Wiley-Interscience, New York, 1990)) and site-directed mutagenesis(Carter, P., in Mutagenesis: A Practical Approach, Chapter 1 (IRL Press,Oxford, UK 1991)). The γ1 isotype was chosen as it has been found to bethe preferred human isotype for supporting ADCC and complement dependentcytotoxicity using matched sets of chimeric (Bruggemann, M. et al., J.Exp. Med. 166:1351-1361 (1987)) or humanized antibodies (Riechmann, L.et al., Nature 332:323-327 (1988)). The PCR-generated V_(L) and V_(H)fragments (FIG. 1) were subsequently mutagenized so that they faithfullyrepresent the sequence of muMAb4D5 determined at the protein level:V_(H) Q1E, V_(L) V104L and T109A (variants are denoted by the amino acidresidue and number followed by the replacement amino acid). The human γ1constant regions are identical to those reported by Ellison et al.(Ellison, J. W. et al., Nucleic Acids Res. 13:4071-4079 (1982)) exceptfor the mutations E359D and M361L (Eu numbering, as in Kabat, E. A. etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987)) which we installed toconvert the antibody from the naturally rare A allotype to the much morecommon non-A allotype (Tramontano, A. et al., J. Mol. Biol. 215:175-182(1990)). This was an attempt to reduce the risk of anti-allotypeantibodies interfering with therapy.

Construction of Humanized Genes. Genes encoding chMAb4D5 light chain andheavy chain Fd fragment (V_(H) and C_(H)1 domains) were subclonedtogether into pUC119 (Vieira, J. & Messing, J., Methods Enzymol.153:3-11 (1987)) to create pAK1 and simultaneously humanized in a singlestep (FIG. 2). Briefly, sets of 6 contiguous oligonucleotides weredesigned to humanize V_(H) and V_(L) (FIG. 1). These oligonucleotidesare 28 to 83 nucleotides in length, contain zero to 19 mismatches to themurine antibody template and are constrained to have 8 or 9 perfectlymatched residues at each end to promote efficient annealing and ligationof adjacent oligonucleotides. The sets of V_(H) and V_(L) humanizationoligonucleotides (5 pmol each) were phosphorylated with either ATP orγ-³²P-ATP (Carter, P. Methods Enzymol. 154:382-403 (1987)) andseparately annealed with 3.7 pmol of pAK1 template in 40 μl 10 mMTris-HCl (pH 8.0) and 10 mM MgCl₂ by cooling from 100° C. to roomtemperature over ˜30 min. The annealed oligonucleotides were joined byincubation with T4 DNA ligase (12 units; New England Biolabs) in thepresence of 2 μl 5 mM ATP and 2 μl 0.1 M DTT for 10 min at 14° C. Afterelectrophoresis on a 6% acrylamide sequencing gel the assembledoligonucleotides were located by autoradiography and recovered byelectroelution. The assembled oligonucleotides (˜0.3 pmol each) weresimultaneously annealed to 0.15 pmol single-strandeddeoxyuridine-containing pAK1 prepared according to Kunkel et al.(Kunkel, T. A. et al., Methods Enzymol. 154:367-382 (1987)) in 10μl 40mM Tris-HCl (pH 7.5) and 16 mM MgCl₂ as above. Heteroduplex DNA wasconstructed by extending the primers with T7 DNA polymerase andtransformed into E. coli BMH 71-18 mutL as previously described (Carter,P., in Mutagenesis: A Practical Approach, Chapter 1 (IRL Press, Oxford,UK 1991)).

The resultant phagemid DNA pool was enriched first for huV_(L) byrestriction purification using XhoI and then for huV_(H) by restrictionselection using StuI as described in Carter, P., in Mutagenesis: APractical Approach, Chapter 1 (IRL Press, Oxford, UK 1991); and inWells, J. A. et al., Phil. Trans. R. Soc. Lond. A 317:415-423 (1986).Resultant clones containing both huV_(L) and huV_(H) genes wereidentified by nucleotide sequencing (Sanger, F. et al., Proc. Natl.Acad. Sci. USA 74:5463-5467 (1977)) and designated pAK2. Additionalhumanized variants were generated by site-directed mutagenesis (Carter,P., in Mutagenesis: A Practical Approach, Chapter 1 (IRL Press, Oxford,UK 1991)). The muMAb4D5 V_(L) and V_(H) gene segments in the transientexpression vectors described above were then precisely replaced withtheir humanized versions.

Expression and Purification of MAb4D5 Variants. Appropriate MAb4D5 lightand heavy chain cDNA expression vectors were co-transfected into anadenovirus transformed human embryonic kidney cell line, 293 (Graham, F.L. et al., J. Gen. virol. 36:59-72 (1977)) using a high efficiencyprocedure (Gorman, C. M. et al., DNA & Prot. Engin. Tech. 2:3-10 (1990);Gorman, C., in DNA Cloning, vol 11, pp 143-190 (D. M. Glover, ed., IRLPress, Oxford, UK 1985)). Media were harvested daily for up to 5 daysand the cells re-fed with serum free media. Antibodies were recoveredfrom the media and affinity purified on protein A sepharose CL-4B(Pharmacia) as described by the manufacturer. The eluted antibody wasbuffer-exchanged into phosphate-buffered saline by G25 gel filtration,concentrated by ultrafiltration (Centriprep-30 or Centricon-100,Amicon), sterile-filtered (Millex-GV, Millipore) and stored at 4° C. Theconcentration of antibody was determined by using both totalimmunoglobulin and antigen binding ELISAs. The standard used washuMAb4D5-5, whose concentration had been determined by amino acidcomposition analysis.

Cell Proliferation Assay. The effect of MAb4D5 variants uponproliferation of the human mammary adenocarcinoma cell line, SK-BR-3,was investigated as previously described (Fendly, B. M. et al., CancerRes. 50:1550-1558 (1990)) using saturating MAb4D5 concentrations.

Affinity Measurements. The antigen binding affinity of MAb4D5 variantswas determined using a secreted form of the p185^(HER2) ECD prepared asdescribed in Fendly, B. M. et al., J. Biol. Resp. Mod. 9:449-455 (1990).Briefly, antibody and p185^(HER2) ECD were incubated in solution untilequilibrium was found to be reached. The concentration of free antibodywas then determined by ELISA using immobilized p185^(HER2) ECD and usedto calculate affinity (K_(d)) according to Friguet et al. (Friguet, B.et al., J. Immunol. Methods 77:305-319 (1985)).

Results

Humanization of muMAb4D5. The muMAb4D5 V_(L) and V_(H) gene segmentswere first cloned by PCR and sequenced (FIG. 1). The variable genes werethen simultaneously humanized by gene conversion mutagenesis usingpreassembled oligonucleotides (FIG. 2). A 311-mer oligonucleotidecontaining 39 mismatches to the template directed 24 simultaneous aminoacid changes required to humanize muMAb4D5 V_(L). Humanization ofmuMAb4D5 V_(H) required 32 amino acid changes which were installed witha 361-mer containing 59 mismatches to the muMAb4D5 template. Two out of8 clones sequenced precisely encode huMAb4D5-5, although one of theseclones contained a single nucleotide imperfection. The 6 other cloneswere essentially humanized but contained a small number of errors: <3nucleotide changes and <1 single nucleotide deletion per kilobase.Additional humanized variants (Table 3) were constructed bysite-directed mutagenesis of huMAb4D5-5.

Expression levels of huMAb4D5 variants were in the range of 7 to 15μg/ml as judged by ELISA using immobilized p185^(HER2) ECD. Successiveharvests of five 10 cm plates allowed 200 μg to 500 mg of each variantto be produced in a week. Antibodies affinity purified on protein A gavea single band on a Coomassie blue stained SDS polyacrylamide gel ofmobility consistent with the expected M_(r) of ˜150 kDa. Electrophoresisunder reducing conditions gave 2 bands consistent with the expectedM_(r) of free heavy (48 kDa) and light (23 kDa) chains (not shown).Amino terminal sequence analysis (10-cycles) gave the mixed sequenceexpected (see FIG. 1) from an equimolar combination of light and heavychains (not shown).

huMAb4D5 Variants. In general, the FR residues were chosen fromconsensus human sequences (Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987)) and CDR residues from muMAb4D5. Additional variants wereconstructed by replacing selected human residues in huMAb4D5-1 withtheir muMAb4D5 counterparts. These are V_(H) residues 71, 73, 78, 93plus 102 and V_(L) residues 55 plus 66 identified by our molecularmodeling. V₁ residue 71 has previously been proposed by others(Tramontano, A. et al., J. Mol. Biol. 215:175-182 (1990)) to be criticalto the conformation of V_(H)-CDR2. Amino acid sequence differencesbetween huMAb4D5 variant molecules are shown in Table 3, together withtheir p185^(HER2) ECD binding affinity and maximal anti-proliferativeactivities against SK-BR-3 cells. Very similar K_(d) values wereobtained for binding of MAb4D5 variants to either SK-BR-3 cells or top185^(HER2) ECD (Table 3). However, K_(d) estimates derived from bindingof MAb4D5 variants to p185^(HER2) ECD were more reproducible withsmaller standard errors and consumed much smaller quantities of antibodythan binding measurements with whole cells.

The most potent humanized variant designed by molecular modeling,huMAb4D5-8, contains 5 FR residues from muMAb4D5. This antibody bindsthe p185^(HER2) ECD 3-fold more tightly than does muMAb4D5 itself (Table3) and has comparable anti-proliferative activity with SK-BR-3 cells(FIG. 3). In contrast, huMAb4D5-1 is the most humanized but least potentmuMAb4D5 variant, created by simply installing the muMAb4D5 CDRs intothe consensus human sequences, huMAb4D5-1 binds the p185^(HER2) ECD80-fold less tightly than does the murine antibody and has no detectableanti-proliferative activity at the highest antibody concentrationinvestigated (16 μg/ml).

The anti-proliferative activity of huMAb4D5 variants against p185^(HER2)overexpressing SK-BR-3 cells is not simply correlated with their bindingaffinity for the p185^(HER2) ECD. For example, installation of threemurine residues into the V_(H) domain of huMAb4D5-2 (D73T, L78A andA93S) to create huMAb4D5-3 does not change the antigen binding affinitybut does confer significant anti-proliferative activity (Table 3).

The importance of V_(H) residue 71 (Tramontano, A. et al., J. Mol Biol.215:175-182 (1990)) is supported by the observed 5-fold increase inaffinity for p185^(HER2) ECD on replacement of R71 in huMAb4D5-1 withthe corresponding murine residue, alanine (huMAb4D5-2). In contrast,replacing V_(H) L78 in huMAb4D54 with the murine residue, alanine(huMAb4D5-5), does not significantly change the affinity for thep185^(HER2) ECD or change anti-proliferative activity, suggesting thatresidue 78 is not of critical functional significance to huMAb4D5 andits ability to interact properly with the extracellular domain ofp185^(HER2).

V_(L) residue 66 is usually a glycine in human and murine κ chainsequences (Kabat, E. A. et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987)) but anarginine occupies this position in the muMAb4D5 k light chain.

The side chain of residue 66 is likely to affect the conformation ofV_(L)-CDR1 and V_(L)-CDR2 and the hairpin turn at 68-69 (FIG. 4).Consistent with the importance of this residue, the mutation V_(L) G66R(huMAb4D5-3→huMAb4D5-5) increases the affinity for the p185^(HER2) ECDby 4-fold with a concomitant increase in anti-proliferative activity.

From molecular modeling it appears that the tyrosyl side chain ofmuMAb4D5 V_(L) residue 55 may either stabilize the conformation ofV_(H)-CDR3 or provide an interaction at the V_(L)-V_(H) interface. Thelatter function may be dependent upon the presence of V_(H) Y102. In thecontext of huMAb4D5-5 the mutations V_(L) E55Y (huMAb4D5-6) and V_(H)V102Y (huMAb4D5-7) individually increase the affinity for p185^(HER2)ECD by 5-fold and 2-fold respectively, whereas together (huMAb4D5-8)they increase the affinity by 11-fold. This is consistent with eitherproposed role of V_(L) Y55 and V_(H) Y102.

Secondary Immune Function of huMAb4D5-8. MuMAb4D5 inhibits the growth ofhuman breast tumor cells which overexpress p185^(HER2) (Hudziak, R. M.et al., Molec. Cell. Biol. 9:1165-1172 (1989)). The antibody, however,does not offer the possibility of direct tumor cytotoxic effects. Thispossibility does arise in huMAb4D5-8 as a result of its high affinity(K_(d)=0.1 μM) and its human IgG₁ subtype. Table 4 compares the ADCCmediated by huMAb4D5-8 with muMAb4D5 on a normal lung epithelial cellline, WI-38, which expresses a low level of p185^(HER2) and on SK-BR-3,which expresses a high level of p185^(HER2). The results demonstratethat: (1) huMAb4D5 has a greatly enhanced ability to carry out ADCC ascompared with its murine parent; and (2) that this activity may beselective for cell types which overexpress p185^(HER2).

Discussion

MuMAb4D5 is potentially useful for human therapy since it is cytostatictowards human breast and ovarian tumor lines overexpressing theHER2-encoded p185^(HER2) receptor-like tyrosine kinase. Since bothbreast and ovarian carcinomas are chronic diseases it is anticipatedthat the optimal MAb4D5 variant molecule for therapy will have lowimmunogenicity and will be cytotoxic rather than solely cytostatic ineffect. Humanization of muMAb4D5 should accomplish these goals. We haveidentified 5 different huMAb4D5 variants which bind tightly top185^(HER2) ECD (K_(d)≦1 nM) and which have significantanti-proliferative activity (Table 3). Furthermore huMAb4D5-8 but notmuMAb4D5 mediates ADCC against human tumor cell lines overexpressingp185^(HER2) in the presence of human effector cells (Table 4) asanticipated for a human γ1 isotype (Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987); Riechmann, L. et al., Nature 332:323-327 (1988)).

Rapid humanization of huMAb4D5 was facilitated by the gene conversionmutagenesis strategy developed here using long preassembledoligonucleotides. This method requires less than half the amount ofsynthetic DNA as does total gene synthesis and does not requireconvenient restriction sites in the target DNA. Our method appears to besimpler and more reliable than a variant protocol recently reported(Rostapshov, V. M. et al., FEBS Lett. 249:379-382 (1989)). Transientexpression of huMAb4D5 in human embryonic kidney 293 cells permitted theisolation of a few hundred micrograms of huMAb4D5 variants for rapidcharacterization by growth inhibition and antigen binding affinityassays. Furthermore, different combinations of light and heavy chainwere readily tested by co-transfection of corresponding cDNA expressionvectors.

The crucial role of molecular modeling in the humanization of muMAb4D5is illustrated by the designed variant huMAb4D5-8 which binds thep185^(HER2) ECD 250-fold more tightly than the simple CDR loop swapvariant, huMAb4D5-1. It has previously been shown that the antigenbinding affinity of a humanized antibody can be increased by mutagenesisbased upon molecular modelling (Riechmann, L. et al., Nature 332:323-327(1988); Queen, C. et al., Proc. Natl. Acad. Sci. USA 86:10029-10033(1989)). Here we have extended this earlier work by others with adesigned humanized antibody which binds its antigen 3-fold more tightlythan the parent rodent antibody. While this result is gratifying,assessment of the success of the molecular modeling must await theoutcome of X-ray structure determination. From analysis of huMAb4D5variants (Table 3) it is apparent that their anti-proliferative activityis not a simple function of their binding affinity for p185^(HER2) ECD.For example the huMAb4D5-8 variant binds p185^(HER2 3)-fold more tightlythan muMAb4D5 but the humanized variant is slightly less potent inblocking the proliferation of SK-BR-3 cells. Additional huMAb4D5variants are currently being constructed in an attempt to identifyresidues triggering the anti-proliferative activity and in an attempt toenhance this activity.

In addition to retaining tight receptor binding and the ability toinhibit cell growth, the huMAb4D5-8 also confers a secondary immunefunction (ADCC). This allows for direct cytotoxic activity of thehumanized molecule in the presence of human effector cells. The apparentselectivity of the cytotoxic activity for cell types which overexpressp185^(HER2) allows for the evolution of a straightforward clinicapproach to those human cancers characterized by overexpression of theHER2 protooncogene.

TABLE 3 p185^(HER2) ECD binding affinity and anti-proliferativeactivities of MAb4D5 variants V_(H) Residue* V_(L) Residue* MAb4D5 cell71 73 78 93 102 55 66 K_(d) ^(†) Relative Variant proliferation^(‡) FR3FR3 FR3 FR3 CDR3 CDR2 FR3 nM huMAb4D5-1 R D L A V E G 25   102 huMAb4D5-2 Ala D L A V E G 4.7 101  huMAb4D5-3 Ala Thr Ala Ser V E G 4.466 huMAb4D5-4 Ala Thr L Ser V E Arg  0.82 56 huMAb4D5-5 Ala Thr Ala SerV E Arg 1.1 48 huMAb4D5-6 Ala Thr Ala Ser V Tyr Arg  0.22 51 huMAb4D5-7Ala Thr Ala Ser Tyr E Arg  0.62 53 huMAb4D5-8 Ala Thr Ala Ser Tyr TyrArg  0.10 54 muMAb4D5 Ala Thr Ala Ser Tyr Tyr Arg  0.30 37 *Human andmurine residues are shown in one letter and three letter amino acid coderespectively. ^(†)K_(d) values for the p185^(HER2) ECD were determinedusing the method of Friguet et al. (43) and the standard error of eachestimate is ≦ ± 10%. ^(‡)Proliferation of SK-BR-3 cells incubated for 96hr with MAb4D5 variants shown as a percentage of the untreated controlas described (Hudziak, R. M. et al., Molec. Cell. Biol. 9:1165-1172(1989)). Data represent the maximal anti-proliferative effect for eachvariant (see FIG. 3A) calculated as the mean of triplicatedeterminations at a MAb4D5 concentration of 8 μg/ml. Data are all takenfrom the same experiment with an estimated standard error of ≦ ± 15%.

TABLE 4 Selectivity of antibody dependent tumor cell cytotoxicitymediated by huMAb4D5-8 Effec- tor: WI-38* SK-BR-3 Target huMAb4D5-huMAb4D5- ratio† muMAb4D5 8 muMAb4D5 8 A.‡   25:1 <1.0 9.3 7.5 40.612.5:1 <1.0 11.1 4.7 36.8 6.25:1 <1.0 8.9 0.9 35.2 3.13:1 <1.0 8.5 4.619.6 B.   25:1 <1.0 3.1 6.1 33.4 12.5:1 <1.0 1.7 5.5 26.2 6.25:1  1.32.2 2.0 21.0 3.13:1 <1.0 0.8 2.4 13.4 *Sensitivity to ADCC of two humancell lines (WI-38, normal lung epithelium; and SK-BR-3, human breasttumor cell line) are compared. WI-38 expresses a low level ofp185^(HER2) (0.6 pg per μg cell protein) and SK-BR-3 expresses a highlevel of p185^(HER2) (64 pg p185^(HER2) per μg cell protein), asdetermined by ELISA (Fendly et al., J. Biol. Resp. Mod. 9:449-455(1990)). †ADCC assays were carried out as described in Brüggeman et al.,J. Exp. Med. 166:1351-1361 (1987). Effector to target ratios were ofIL-2 activated human peripheral blood lymphocytes to either WI-38fibroblasts or SK-BR-3 tumor cells in 96-well microtiter plates for 4hours at 37° C. Values given represent percent specific cell lysis asdetermined by ⁵¹Cr release. Estimated standard error in thesequadruplicate determinations was ≦ ±10%. ‡Monoclonal antibodyconcentrations used were 0.1 μg/ml (A) and 0.1 μg/ml (B).

* Sensitivity to ADCC of two human cell lines (WI-38, normal lungepithelium; and SK-BR-3, human breast tumor cell line) are compared.WI-38 expresses a low level of p185^(HER2) (0.6 pg per μg cell protein)and SK-BR-3 expresses a high level of p185^(HER2) (64 pg p185^(HER2) perμg cell protein), as determined by ELISA (Fendly et al., J. Biol. Resp.Mod. 9:449-455 (1990)).

† ADCC assays were carried out as described in Bruggemann et al., J.Exp. Med. 166:1351-1361 (1987). Effector to target ratios were of IL-2activated human peripheral blood lymphocytes to either WI-38 fibroblastsor SK-BR-3 tumor cells in 96-well microtiter plates for 4 hours at 37°C. Values given represent percent specific cell lysis as determined by⁵¹Cr release. Estimated standard error in these quadruplicatedeterminations was ≦±10%.

‡ Monoclonal antibody concentrations used were 0.1 μg/ml (A) and 0.1μg/ml (B).

Example 2 Schematic Method for Humanizing an Antibody Sequence

This example illustrates one stepwise elaboration of the methods forcreating a humanized sequence described above. It will be understoodthat not all of these steps are essential to the claimed invention, andthat steps may be taken in different order.

1. ascertain a consensus human variable domain amino acid sequence andprepare from it a consensus structural model.

2. prepare model of import (the non-human domain to be humanized)variable domain sequences and note structural differences with respectto consensus human model.

3. identify CDR sequences in human and in import, both by using Kabat(supra, 1987) and crystal structure criteria. If there is any differencein CDR identity from the different criteria, use of crystal structuredefinition of the CDR, but retain the Kabat residues as importantframework residues to import.

4. substitute import CDR sequences for human CDR sequences to obtaininitial “humanized” sequence.

5. compare import non-CDR variable domain sequence to the humanizedsequence and note divergences.

6. Proceed through the following analysis for each amino acid residuewhere the import diverges from the humanized.

a. If the humanized residue represents a residue which is generallyhighly conserved across all species, use the residue in the humanizedsequence. If the residue is not conserved across all species, proceedwith the analysis described in 6b.

b. If the residue is not generally conserved across all species, ask ifthe residue is generally conserved in humans.

i. If the residue is generally conserved in humans but the importresidue differs, examine the structural models of the import and humansequences and determine if the import residue would be likely to affectthe binding or biological activity of the CDRs by considering 1) couldit bind antigen directly and 2) could it affect the conformation of theCDR.

If the conclusion is that an affect on the CDRs is likely, substitutethe import residue. If the conclusion is that a CDR affect is unlikely,leave the humanized residue unchanged.

ii. If the residue is also not generally conserved in humans, examinethe structural models of the import and human sequences and determine ifthe import residue would be likely to affect the binding or biologicalactivity of the CDRs be considering 1) could it bind antigen directlyand 2) could it affect the conformation of the CDR. If the conclusion isthat an affect on the CDRs is likely, substitute the import residue. Ifthe conclusion is that a CDR affect is unlikely, proceed to the nextstep.

a) Examine the structural models of the import and human sequences anddetermine if the residue is exposed on the surface of the domain or isburied within. If the residue is exposed, use the residue in thehumanized sequence. If the residue is buried, proceed to the next step.

(i) Examine the structural models of the import and human sequences anddetermine if the residue is likely to affect the V_(L)-V_(H) interface.Residues involved with the interface include: 34L, 36L, 38L, 43L, 33L,36L, 85L, 87L, 89L, 91L, 96L, 98L, 35H, 37H, 39H, 43H, 45H, 47H, 60H,91H, 93H, 95H, 100H, and 103H. If no effect is likely, use the residuein the humanized sequence. If some affect is likely, substitute theimport residue.

7. Search the import sequence, the consensus sequence and the humanizedsequence for glycosylation sites outside the CDRs, and determine if thisglycosylation site is likely to have any affect on antigen bindingand/or biological activity. If no effect is likely, use the humansequence at that site; if some affect is likely, eliminate theglycosylation site or use the import sequence at that site.

8. After completing the above analysis, determine the planned humanizedsequence and prepare and test a sample. If the sample does not bind wellto the target antigen, examine the particular residues listed below,regardless of the question of residue identity between the import andhumanized residues.

a. Examine particular peripheral (non-CDR) variable domain residues thatmay, due to their position, possibly interact directly with amacromolecular antigen, including the following residues (where the *indicates residues which have been found to interact with antigen basedon crystal structures):

i. Variable light domain: 36, 46, 49*, 63-70

ii. Variable heavy domain: 2, 47*, 68, 70, 73-76.

b. Examine particular variable domain residues which could interactwith, or otherwise affect, the conformation of variable domain CDRs,including the following (not including CDR residues themselves, since itis assumed that, because the CDRs interact with one another, any residuein one CDR could potentially affect the conformation of another CDRresidue) (L=LIGHT, H=HEAVY, residues appearing in bold are indicated tobe structurally important according the Chothia et al., Nature 342:877(1989), and residues appearing in italic were altered duringhumanization by Queen et al. (PDL), Proc. Natl. Acad. Sci. USA 86:10029(1989) and Proc. Natl. Acad. Sci. USA 88:2869 (1991).):

i. Variable light domain:

a) CDR-1 (residues 24L-34L): 2L, 4L, 66L-69L, 71L

b) CDR-2 (residues 50L-56L): 35L, 46L, 47L, 48L, 49L, 58L, 62L, 64L-66L,71L, 73L

c) CDR-3 (residues 89L-97L): 2L, 4L, 36L, 98L, 37H, 45H, 47H, 58H, 60H

ii. Variable heavy domain:

a) CDR-1 (residues 26H-35H): 2H, 4H, 24H, 36H, 71H, 73H, 76H, 78H, 92H,94H

b) CDR-2 (residues 5OH-55H): 49H, 69H, 69H, 71H, 73H, 78H

c) CDR-3 (residues 95H-102H): examine all residues as possibleinteraction partners with this loop, because this loop varies in sizeand conformation much more than the other CDRs.

9. If after step 8 the humanized variable domain still is lacking indesired binding, repeat step 8. In addition, re-investigate any buriedresidues which might affect the V_(L)-V_(H) interface (but which wouldnot directly affect CDR conformation). Additionally, evaluate theaccessibility of non-CDR residues to solvent.

Example 3 Engineering a Humanized Bispecific F(ab′)₂ Fragment

This example demonstrates the construction of a humanized bispecificantibody (BsF(ab′)₂v1 by separate E. coli expression of each Fab′ armfollowed by directed chemical coupling in vitro. BsF(ab′)₂ v1(anti-CD3/anti-p185^(HER2)) was demonstrated to retarget the cytotoxicactivity of human CD3⁺ CTL in vitro against the human breast tumor cellline, SK-BR-3, which overexpresses the p185^(HER2) product of theprotooncogene HER2. This example demonstrates the minimalistichumanization strategy of installing as few murine residues as possibleinto a human antibody in order to recruit antigen-binding affinity andbiological properties comparable to that of the murine parent antibody.This strategy proved very successful for the anti-p185^(HER2)arm ofBsF(ab′)₂v1. In contrast BsF(ab′)₂ v1 binds to T cells via its anti-CD3arm much less efficiently than does the chimeric BsF(ab′)₂ whichcontains the variable domains of the murine parent anti-CD3 antibody.Here we have constructed additional BsF(ab′)₂ fragments containingvariant anti-CD3 arms with selected murine residues restored in anattempt to improve antibody binding to T cells. One such variant,BsF(ab′)₂ v9, was created by replacing six residues in the secondhypervariable loop of the anti-CD3 heavy chain variable domain ofBsF(ab′)₂ v1 with their counterparts from the murine parent anti-CD3antibody. BsF(ab′)₂ v9 binds to T cells (Jurkat) much more efficientlythan does BsF(ab′)₂ v1 and almost as efficiently as the chimericBsF(ab′)₂. This improvement in the efficiency of T cell binding of thehumanized BsF(ab′)₂ is an important step in its development as apotential therapeutic agent for the treatment ofp185^(HER2)-overexpressing cancers.

Bispecific antibodies (BsAbs) with specificities for tumor-associatedantigens and surface markers on immune effector cells have provedeffective for retargeting effector cells to kill tumor targets both invitro and in vivo (reviewed by Fanger, M. W. et al., Immunol. Today 10:92-99 (1989): Fanger, M. W. et al., Immunol. Today 12: 51-54 (1991); andNelson, H., Cancer Cells 3: 163-172(1991)). BsF(ab′)₂ fragments haveoften been used in preference to intact BsAbs in retargeted cellularcytotoxicity to avoid the risk of killing innocent bystander cellsbinding to the Fc region of the antibody. An additional advantage ofBsF(ab′)₂ over intact BsAbs is that they are generally much simpler toprepare free of contaminating monospecific molecules (reviewed bySongsivilai, S. and Lachmann, P. J., Clin. Exp. Immunol. 79: 315-321(1990) and Nolan, O. and O'Kennedy, R., Biochim. Biophys. Acta 1040:1-11 (1990)).

BsF(ab′)₂ fragments are traditionally constructed by directed chemicalcoupling of Fab′ fragments obtained by limited proteolysis plus mildreduction of the parent rodent monoclonal Ab (Brennan, M. et al.,Science 229, 81-83 (1985) and Glennie, M. J. et al., J. Immunol. 139:2367-2375 (1987)). One such BsF(ab′)₂ fragment (anti-glioma associatedantigen/anti-CD3) was found to have clinical efficacy in glioma patients(Nitta, T. et al., Lancet 335: 368-371 (1990) and another BsF(ab′)₂(anti-indium chelate/anti-carcinoembryonic antigen) allowed clinicalimaging of colorectal carcinoma (Stickney, D. R. et al., Antibody,Immunoconj. Radiopharm. 2:1-13 (1989)). Future BsF(ab′)₂ destined forclinical applications are likely to be constructed from antibodies whichare either human or at least “humanized” (Riechmann, L. et al., Nature332: 323-327 (1988) to reduce their immunogenicity (Hale, G. et al.,Lancet i: 1394-1399 (1988)).

Recently a facile route to a fully humanized BsF(ab′)₂ fragment designedfor tumor immunotherapy has been demonstrated (Shalaby, M. R. et al., J.Exp. Med. 175: 217-225 (1992)). This approach involves separate E. coliexpression of each Fab′ arm followed by traditional directed chemicalcoupling in vitro to form the BsF(ab′)₂. One arm of the BsF(ab′)₂ was ahumanized version (Carter, P. et al., Proc. Natl. Acad. Sci. USA (1992a)and Carter, P., et al., Bio/Technology 10: 163-167 (1992b)) of themurine monoclonal Ab 4D5 which is directed against the p185^(HER2)product of the protooncogene HER2 (c-erbB-2) (Fendly, B. M. et al.Cancer Res. 50: 1550-1558 (1989)). The humanization of the antibody 4D5is shown in Example 1 of this application. The second arm was aminimalistically humanized anti-CD3 antibody (Shalaby et al. supra)which was created by installing the CDR loops from the variable domainsof the murine parent monoclonal Ab UCHT1 (Beverley, P. C. L. andCallard, R. E., Eur. J. Immunol. 11: 329-334 (1981)) into the humanizedanti-p185^(HER2) antibody. The BsF(ab′)₂ fragment containing the mostpotent humanized anti-CD3 variant (v1) was demonstrated by flowcytometry to bind specifically to a tumor target overexpressingp185^(HER2) and to human peripheral blood mononuclear cells carryingCD3. In addition, BsF(ab′)₂ v1 enhanced the cytotoxic effects ofactivated human CTL 4-fold against SK-BR-3 tumor cells overexpressingp185^(HER2). The example descries efforts to improve the antigen bindingaffinity of the humanized anti-CD3 arm by the judicious recruitment of asmall number of additional murine residues into the minimalisticallyhumanized anti-CD3 variable domains.

Materials and Methods

Construction of Mutations in the Anti-CD3 Variable Region Genes

The construction of genes encoding humanized anti-CD3 variant 1 (v1)variable light (V_(L)) and heavy (V_(H)) chain domains in phagemidpUC119 has been described (Shalaby et al. supra). Additional anti-CD3variants were generated using an efficient site-directed mutagenesismethod (Carter, P., Mutagenesis: a practical approach, (M. J. McPherson,Ed.), Chapter 1, IRL Press, Oxford, UK (1991)) using mismatchedoligonucleotides which either install or remove unique restrictionsites. Oligonucleotides used are listed below using lowercase toindicate the targeted mutations. Corresponding coding changes aredenoted by the starting amino acid in one letter code followed by theresidue numbered according to Kabat, E. A. et al., Sequences of Proteinsof Immunological Interest, 5^(th) edition, National Institutes ofHealth, Bethesda, Md., USA (1991), then the replacement amino acid andfinally the identity of the anti-CD3 variant:

HX11, 5′ GTAGATAAATCCtctAACACAGCCTAtCTGCAAATG 3′ (SEQ.ID. NO. 11) V_(H)K75S, v6;

HX12, 5′ GTAGATAAATCCAAAtctACAGCCTAtCTGCAAATG 3′ (SEQ.ID. NO. 12) V_(H)N76S, v7;

HX13, 5′ GTAGATAAATCCtcttctACAGCCTAtCTGCAAATG 3′ (SEQ.ID. NO. 13) V_(H)K75S:N76S, v8;

X14, 5° C.TTATAAAGGTGTTtCcACCTATaaCcAgAaatTCAAGGatCGTTTCACgATAtcCGTAGATAAATCC 3′ (SEQ.ID.NO. 14) V_(H) T57S:A60N:D61Q:S62K:V63F:G65D, v9;

LX6, 5° C.TATACCTCCCGTCTgcatTCTGGAGTCCC 3′ (SEQ.ID. NO. 15) V_(L) E55H,v11. Oligonucleotides HX11, HX12 and HX13 each remove a site for BspMI,whereas LX6 removes a site for XhoI and HX14 installs a site for EcoRV(bold). Anti-CD3 variant v10 was constructed from v9 by site-directedmutagenesis using oligonucleotide HX13. Mutants were verified bydideoxynucleotide sequencing (Sanger, F. et al., Proc. Natl. Acad. Sci.USA 74: 5463-5467 (1977)).

E. coli Expression of Fab′ Fragments

The expression plasmid, pAK19, for the co-secretion of light chain andheavy chain Fd′ fragment of the most preferred humanizedanti-p185^(HER2) variant, HuMAb4D5-8, is described in Carter et al.,1992b, supra. Briefly, the Fab′ expression unit is bicistronic with bothchains under the transcriptional control of the phoA promoter. Genesencoding humanized V_(L) and V_(H) domains are precisely fused on their5′ side to a gene segment encoding the heat-stable enterotoxin 11 signalsequence and on their 3′ side to human k, C_(L) and IgG1 CH1 constantdomain genes, respectively. The C_(H)1 gene is immediately followed by asequence encoding the hinge sequence CysAlaAla and followed by abacteriophage λ t₀ transcriptional terminator.

Fab′ expression plasmids for chimeric and humanized anti-CD3 variants(v1 to v4, Shalaby et al., supra; v6 to v12, this study) were createdfrom pAK19 by precisely replacing anti-p185^(HER2) V_(L) and V_(H) genesegments with those encoding murine and corresponding humanized variantsof the anti-CD3 antibody, respectively, by sub-cloning and site-directedmutagenesis. The Fab′ expression plasmid for the most potent humanizedanti-CD3 variant identified in this study (v9) is designated pAK22. Theanti-p185^(HER2) Fab′ fragment was secreted from E. coli K12 strain 25F2containing plasmid pAK19 grown for 32 to 40 hr at 37° C. in an aerated10 liter fermentor. The final cell density was 120-150 OD₅₅₀ and thetiter of soluble and functional anti-p185^(HER2) Fab′ was 1-2 g/liter asjudged by antigen binding ELISA (Carter et al., 1992b, supra). Anti-CD3Fab′ variants were secreted from E. coli containing correspondingexpression plasmids using very similar fermentation protocols. Thehighest expression titers of chimeric and humanized anti-CD3 variantswere 200 mg/liter and 700 mg/liter, respectively, as judged by totalimmunoglobulin ELISA.

Construction of BsF(ab′)₂ Fragments

Fab′ fragments were directly recovered from E. coli fermentation pastesin the free thiol form (Fab′-SH) by affinity purification onStreptococcal protein G at pH 5 in the presence of EDTA (Carter et al.,1992b supra). Thioether linked BsF(ab′)₂ fragments(anti-p185^(HER2)/anti-CD3) were constructed by the procedure of Glennieet al. supra with the following modifications. Anti-p185^(HER2) Fab′-SHin 100 mM Tris acetate, 5 mM EDTA (pH 5.0) was reacted with 0.1 vol of40 mM N,N′-1,2-phenylenedimalemide (o-PDM) in dimethyl formamide for˜1.5 hr at 20° C. Excess o-PDM was removed by protein G purification ofthe Fab′ maleimide derivative (Fab′-mal) followed by buffer exchangeinto 20 mM sodium acetate, 5 mM EDTA (pH 5.3) (coupling buffer) usingcentriprep-30 concentrators (Amicon). The total concentration of Fab′variants was estimated from the measured absorbance at 280 nm(HuMAb4D5-8 Fab′ e^(0.1%)=1.56, Carter et al., 1992b, supra). The freethiol content of Fab′ preparations was estimated by reaction with5,5′-dithiobis(2-nitrobenzoic acid) as described by Creighton, T. E.,Protein structure: a practical approach, (T. E. Creighton, Ed.), Chapter7, IRL Press, Oxford, UK (1990). Equimolar amounts of anti-p185^(HER2)Fab′-mal (assuming quantitative reaction of Fab′-SH with o-PDM) and eachanti-CD3 Fab′-SH variant were coupled together at a combinedconcentration of 1 to 2.5 mg/ml in the coupling buffer for 14 to 48 hrat 4° C. The coupling reaction was adjusted to 4 mM cysteine at pH 7.0and incubated for 15 min at 20° C. to reduce any unwanteddisulfide-linked F(ab′)₂ formed. These reduction conditions aresufficient to reduce inter-heavy chain disulfide bonds with virtually noreduction of the disulfide between light and heavy chains. Any freethiols generated were then blocked with 50 mM iodoacetamide. BsF(ab′)₂was isolated from the coupling reaction by S100-HR (Pharmacia) sizeexclusion chromatography (2.5 cm×100 cm) in the presence of PBS. TheBsF(ab′)₂ samples were passed through a 0.2 mm filter flash frozen inliquid nitrogen and stored at −70° C.

Flow Cytometric Analysis of F(ab′)₂ Binding to Jurkat Cells

The Jurkat human acute T cell leukemia cell line was purchased from theAmerican Type Culture Collection Manassas, Va. (ATCC TIB 152) and grownas recommended by the ATCC. Aliquots of 10⁶ Jurkat cells were incubatedwith appropriate concentrations of BsF(ab′)₂ (anti-p185^(HER2)/anti-CD3variant) or control mono-specific anti-p185^(HER2) F(ab′)₂ in PBS plus0.1% (w/v) bovine serum albumin and 10 mM sodium azide for 45 min at 4°C. The cells were washed and then incubated with fluorescein-conjugatedgoat anti-human F(ab′)₂ (Organon Teknika, West Chester, Pa.) for 45 minat 4° C. Cells were washed and analyzed on a FACScan® (Becton Dickinsonand Co., Mountain View, Calif.). Cells (8×10³) were acquired by listmode and gated by forward light scatter versus side light scatterexcluding dead cells and debris.

Results

Design of Humanized Anti-CD3 Bariants

The most potent humanized anti-CD3 variant previously identified, v1,differs from the murine parent antibody, UCHT1 at 19 out of 107 aminoacid residues within V_(L) and at 37 out of 122 positions within V_(H)(Shalaby et al., supra) 1992). Here we recruited back additional murineresidues into anti-CD3 v1 in an attempt to improve the binding affinityfor CD3. The strategy chosen was a compromise between minimizing boththe number of additional murine residues recruited and the number ofanti-CD3 variants to be analyzed. We focused our attentions on a few CDRresidues which were originally kept as human sequences in ourminimalistic humanization regime. Thus human residues in V_(H) CDR2 ofanti-CD3 v1 were replaced en bloc with their murine counterparts to giveanti-CD3 v9: T57S:A60N:D61Q:S62K:V63F:G65D(SEQ ID NO. 20). Similarly,the human residue E55 in V_(L) CDR2 of anti-CD3 v1 was replaced withhistidine from the murine anti-CD3 antibody to generate anti-CD3 v11. Inaddition, V_(H) framework region (FR) residues 75 and 76 in anti-CD3 v1were also replaced with their murine counterparts to create anti-CD3 v8:K75S:N76S. V_(H) residues 75 and 76 are located in a loop close to V_(H)CDR1 and CDR2 and therefore might influence antigen binding. Additionalvariants created by combining mutations at these three sites aredescribed below.

Preparation of BsF(ab′)₂ Fragments

Soluble and functional anti-p185^(HER2) and anti-CD3 Fab′ fragments wererecovered directly from corresponding E. coli fermentation pastes withthe single hinge cysteine predominantly in the free thiol form (75-100%Fab′-SH) by affinity purification on Streptococcal protein G at pH 5 inthe presence of EDTA (Carter et al., 1992b, supra). Thioether-linkedBsF(ab′)₂ fragments were then constructed by directed coupling usingo-PDM as described by Glennie et al., supra. One arm was always the mostpotent humanized anti-p185^(HER2) variant, HuMAb4D5-8 (Carter et al.,1992a, supra) and the other either a chimeric or humanized variant ofthe anti-CD3 antibody. Anti-p185^(HER2) Fab′-SH was reacted with o-PDMto form the maleimide derivative (Fab′-mal) and then coupled to theFab′-SH for each anti-CD3 variant. F(ab′)₂ was then purified away fromunreacted Fab′ by size exclusion chromatography as shown for arepresentative preparation (BsF(ab′)₂ v8) in data not shown. The F(ab′)₂fragment represents ˜54% of the total amount of antibody fragments (bymass) as judged by integration of the chromatograph peaks.

SDS-PAGE analysis of this BsF(ab′)₂ v8 preparation under non-reducingconditions gave one major band with the expected mobility (M_(t)˜96 kD)as well as several very minor bands (data not shown). Amino-terminalsequence analysis of the major band after electroblotting on topolyvinylidene difluoride membrane Matsudaira, P., J. Biol. Chem. 262:10035-10038 (1987) gave the expected mixed sequence from astoichiometric 1:1 mixture of light and heavy chains (V_(L)/V_(H): D/E,I/V, Q/Q, M/L, T/V, O/E, S/S) expected for BsF(ab′)₂. The amino terminalregion of both light chains are identical as are both heavy chains andcorrespond to consensus human FR sequences. We have previouslydemonstrated that F(ab′)₂ constructed by directed chemical couplingcarry both anti-p185^(HER2) and anti-CD3 antigen specificities (Shalabyet al., supra). The level of contamination of the BsF(ab′)₂ withmonospecific F(ab′)₂ is likely to be very low since mock couplingreactions with either anti-p185 Fab′-mal or anti-CD3 Fab′-SH alone didnot yield detectable quantities of F(ab′)₂ Furthermore the couplingreaction was subjected to a mild reduction step followed by alkylationto remove trace amounts of disulfide-linked F(ab′)₂ that might bepresent. SDS-PAGE of the purified F(ab′)₂ under reducing conditions gavetwo major bands with electrophoretic mobility and amino terminalsequence anticipated for free light chain and thioether-linked heavychain dimers.

Scanning LASER densitometry of a o-PDM coupled F(ab′)₂ preparationsuggest that the minor species together represent ˜10% of the protein.These minor contaminants were characterized by amino terminal sequenceanalysis and were tentatively identified on the basis of stoichiometryof light and heavy chain sequences and their electrophoretic mobility(data not shown). These data are consistent with the minor contaminantsincluding imperfect F(ab′)₂ in which the disulfide bond between lightand heavy chains is missing in one or both arms, trace amounts of Fab′and heavy chain thioether-linked to light chain.

Binding of BsF(ab′)₂ to Jurkat Cells

Binding of BsF(ab′)₂ containing different anti-CD3 variants to Jurkatcells (human acute T cell leukemia) was investigated by flow cytometry(data not shown). BsF(ab′)₂ v9 binds much more efficiently to Jurkatcells than does our starting molecule, BsF(ab′)₂ v1, and almost asefficiently as the chimeric BsF(ab′)₂. Installation of additional murineresidues into anti-CD3 v9 to create v10 (V_(H) K75S:N76S) and v12 (V_(H)K75S:N76S plus V_(L) E55H) did not further improve binding ofcorresponding BsF(ab′)₂ to Jurkat cells. Nor did recruitment of thesemurine residues into anti-CD3 v1 improve Jurkat binding: V_(H)K7⁵S (v6),V_(H)N76S (v7), V_(H) K75S:N76S (v8), V_(L) E55H (v11) (not shown).BsF(ab′)₂ v9 was chosen for future study since it is amongst the mostefficient variants in binding to Jurkat cells and contains fewest murineresidues in the humanized anti-CD3 arm. A monospecific anti-p185^(HER2)F(ab′)₂ did not show significant binding to Jurkat cells consistent withthe interaction being mediated through the anti-CD3 arm.

Discussion

A minimalistic strategy was chosen to humanize the anti-p185^(HER2)(Carter et al., 1992a, supra) and anti-CD3 arms (Shalaby et al., supra)of the BsF(ab′)₂ in this study in an attempt to minimize the potentialimmunogenicity of the resulting humanized antibody in the clinic. Thuswe tried to install the minimum number of murine CDR and FR residuesinto the context of consensus human variable domain sequences asrequired to recruit antigen-binding affinity and biological propertiescomparable to the murine parent antibody. Molecular modeling was usedfirstly to predict the murine FR residues which might be important toantigen binding and secondly to predict the murine CDR residues thatmight not be required. A small number of humanized variants were thenconstructed to test these predictions.

Our humanization strategy was very successful for the anti-p185^(HER2)antibody where one out of eight humanized variants (HuMAb4D5-8, IgG1)was identified that bound the p185^(HER2) antigen ˜3-fold more tightlythan the parent murine antibody (Carter et al., 1992a, supra).HuMAb4D5-8 contains a total of five murine FR residues and nine murineCDR residues, including V_(H) CDR2 residues 60-65, were discarded infavor of human counterparts. In contrast, BsF(ab′)₂ v1 containing themost potent humanized anti-CD3 variant out of four originallyconstructed (Shalaby et al., supra) binds J6 cells with an affinity(K_(d)) of 140 nM which is ˜70-fold weaker than that of thecorresponding chimeric BsF(ab′)₂.

Here we have restored T cell binding of the humanized anti-CD3 close tothat of the chimeric variant by replacing six human residues in V_(H)CDR2 with their murine counterparts: T57S:A60N:D61 Q:S62K:V63F:G65D(anti-CD3 v9, FIG. 5). It appears more likely that these murine residuesenhance antigen binding indirectly by influencing the conformation ofresidues in the N-terminal part of V_(H) CDR2 rather than by directlycontacting antigen. Firstly, only N-terminal residues in V_(H) CDR2(50-58) have been found to contact antigen in one or more of eightcrystallographic structures of antibody/antigen complexes (Kabat et al.,supra; and Mian, I. S. et al., J. Mol. Biol. 217: 133-151 (1991), FIG.5). Secondly, molecular modeling suggests that residues in theC-terminal part of V_(H) CDR2 are at least partially buried (FIG. 5).BsF(ab′)₂ v9 binds to SK-BR-3 breast tumor cells with equal efficiencyto BsF(ab′)₂ v1 and chimeric BsF(ab′)₂ as anticipated since theanti-p185^(HER2) arm is identical in all of these molecules (Shalaby etal., supra, not shown).

Our novel approach to the construction of BsF(ab′)₂ fragments exploitsan E. coli expression system which secretes humanized Fab′ fragments atgram per liter titers and permits their direct recovery as Fab′-SH(Carter et al., 1992b, supra). Traditional directed chemical coupling ofFab′-SH fragments is then used to form BsF(ab′)₂ in vitro (Brennan etal., supra; and Glennie et al., supra). This route to Fab′-SH obviatesproblems which are inherent in their generation from intact antibodies:differences in susceptibility to proteolysis and nonspecific cleavageresulting in heterogeneity, low yield as well as partial reduction thatis not completely selective for the hinge disulfide bonds. The strategyof using E. coli-derived Fab′-SH containing a single hinge cysteineabolishes some sources of heterogeneity in BsF(ab′)₂ preparation such asintra-hinge disulfide formation and contamination with intact parentantibody whilst greatly diminishes others, e.g. formation of F(ab′)₃fragments.

BsF(ab′) 2 fragments constructed here were thioether-linked asoriginally described by Glennie et al., supra with future in vivotesting of these molecules in mind. Thioether bonds, unlike disulfidebonds, are not susceptible to cleavage by trace amounts of thiol, whichled to the proposal that thioether-linked Flab′)₂ may be more stablethan disulfide-linked F(ab′)₂ in vivo (Glennie et al., supra). Thishypothesis is supported by our preliminary pharmacokinetic experimentsin normal mice which suggest that thioether-linked BsF(ab′)₂ v1 has a3-fold longer plasma residence time than BsF(ab′)₂ v1 linked by a singledisulfide bond. Disulfide and thioether-linked chimeric BsF(ab′)₂ werefound to be indistinguishable in their efficiency of cell binding and intheir retargeting of CTL cytotoxicity, which suggests that o-PDMdirected coupling does not compromise binding of the BsF(ab′)₂ to eitherantigen (not shown). Nevertheless the nature of the linkage appears notto be critical since a disulfide-linked BsF(ab′)₂ (murineanti-p185^(HER2)/murine anti-CD3) was recently shown by others(Nishimura et al., Int. J. Cancer 50: 800-804 (1992) to have potentanti-tumor activity in nude mice. Our previous study (Shalaby et al.,supra) together with this one and that of Nishimura, T. et al., supraimprove the potential for using BsF(ab′)₂ in targeted immunotherapy ofp185^(HER2)-overexpressing cancers in humans.

Example 4 Humanization of an Anti-CD18 Antibody

A murine antibody directed against the leukocyte adhesion receptorβ-chain (known as the H52 antibody) was humanized following the methodsdescribed above. FIGS. 6A and 6B provide amino acid sequence comparisonsfor the murine and humanized antibody light chains and heavy chains.

26 109 amino acids Amino Acid Linear 1 Asp Ile Gln Met Thr Gln Ser ProSer Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr CysArg Ala Ser Gln Asp Val Asn 20 25 30 Thr Ala Val Ala Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Phe Leu GluSer Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp PheThr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr TyrCys Gln Gln 80 85 90 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr LysVal Glu 95 100 105 Ile Lys Arg Thr 120 amino acids Amino Acid Linear 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30 AspThr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu TrpVal Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 50 55 60 Ala Asp SerVal Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75 Lys Asn Thr AlaTyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr TyrCys Ser Arg Trp Gly Gly Asp Gly Phe Tyr 95 100 105 Ala Met Asp Val TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 120 109 amino acidsAmino Acid Linear 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln AspVal Ser 20 25 30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 6570 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 8590 Tyr Asn Ser Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100105 Ile Lys Arg Thr 120 amino acids Amino Acid Linear 4 Glu Val Gln LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Asp Tyr Ala Met SerTrp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Val IleSer Glu Asn Gly Ser Asp Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln MetAsn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg AspArg Gly Gly Ala Val Ser 95 100 105 Tyr Phe Asp Val Trp Gly Gln Gly ThrLeu Val Thr Val Ser Ser 110 115 120 109 amino acids Amino Acid Linear 5Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val 1 5 10 15Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asn 20 25 30 ThrAla Val Ala Trp Tyr Gln Gln Lys Pro Gly His Ser Pro Lys 35 40 45 Leu LeuIle Tyr Ser Ala Ser Phe Arg Tyr Thr Gly Val Pro Asp 50 55 60 Arg Phe ThrGly Asn Arg Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val GlnAla Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 His Tyr Thr Thr ProPro Thr Phe Gly Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys Arg Ala 120amino acids Amino Acid Linear 6 Glu Val Gln Leu Gln Gln Ser Gly Pro GluLeu Val Lys Pro Gly 1 5 10 15 Ala Ser Leu Lys Leu Ser Cys Thr Ala SerGly Phe Asn Ile Lys 20 25 30 Asp Thr Tyr Ile His Trp Val Lys Gln Arg ProGlu Gln Gly Leu 35 40 45 Glu Trp Ile Gly Arg Ile Tyr Pro Thr Asn Gly TyrThr Arg Tyr 50 55 60 Asp Pro Lys Phe Gln Asp Lys Ala Thr Ile Thr Ala AspThr Ser 65 70 75 Ser Asn Thr Ala Tyr Leu Gln Val Ser Arg Leu Thr Ser GluAsp 80 85 90 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr95 100 105 Ala Met Asp Tyr Trp Gly Gln Gly Ala Ser Val Thr Val Ser Ser110 115 120 27 base pairs Nucleic Acid Single Linear 7 TCCGATATCCAGCTGACCCA GTCTCCA 27 31 base pairs Nucleic Acid Single Linear 8GTTTGATCTC CAGCTTGGTA CCHSCDCCGA A 31 22 base pairs Nucleic Acid SingleLinear 9 AGGTSMARCT GCAGSAGTCW GG 22 34 base pairs Nucleic Acid SingleLinear 10 TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG 34 36 base pairs NucleicAcid Single Linear 11 GTAGATAAAT CCTCTAACAC AGCCTATCTG CAAATG 36 36 basepairs Nucleic Acid Single Linear 12 GTAGATAAAT CCAAATCTAC AGCCTATCTGCAAATG 36 36 base pairs Nucleic Acid Single Linear 13 GTAGATAAATCCTCTTCTAC AGCCTATCTG CAAATG 36 68 base pairs Nucleic Acid Single Linear14 CTTATAAAGG TGTTTCCACC TATAACCAGA AATTCAAGGA TCGTTTCACG 50 ATATCCGTAGATAAATCC 68 30 base pairs Nucleic Acid Single Linear 15 CTATACCTCCCGTCTGCATT CTGGAGTCCC 30 107 amino acids Amino Acid Linear 16 Asp IleGln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu 1 5 10 15 Gly AspArg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg 20 25 30 Asn Tyr LeuAsn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 35 40 45 Leu Leu Ile TyrTyr Thr Ser Arg Leu His Ser Gly Val Pro Ser 50 55 60 Lys Phe Ser Gly SerGly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 65 70 75 Ser Asn Leu Glu Gln GluAsp Ile Ala Thr Tyr Phe Cys Gln Gln 80 85 90 Gly Asn Thr Leu Pro Trp ThrPhe Ala Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys 107 amino acids AminoAcid Linear 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala SerVal 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp IleArg 20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys35 40 45 Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser 5055 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 65 7075 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys 107 amino acids Amino Acid Linear 18 Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile ThrCys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ala Ala Ser Ser LeuGlu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr TyrTyr Cys Gln Gln 80 85 90 Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly ThrLys Val Glu 95 100 105 Ile Lys 122 amino acids Amino Acid Linear 19 GluVal Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 1 5 10 15 AlaSer Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 Gly TyrThr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu 35 40 45 Glu Trp MetGly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr 50 55 60 Asn Gln Lys PheLys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser 65 70 75 Ser Ser Thr Ala TyrMet Glu Leu Leu Ser Leu Thr Ser Glu Asp 80 85 90 Ser Ala Val Tyr Tyr CysAla Arg Ser Gly Tyr Tyr Gly Asp Ser 95 100 105 Asp Trp Tyr Phe Asp ValTrp Gly Ala Gly Thr Thr Val Thr Val 110 115 120 Ser Ser 122 amino acidsAmino Acid Linear 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val GlnPro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr SerPhe Thr 20 25 30 Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys GlyLeu 35 40 45 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr50 55 60 Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser 6570 75 Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 8590 Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser 95 100105 Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115120 Ser Ser 122 amino acids Amino Acid Linear 21 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Ala Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Val Ile Ser GlyAsp Gly Gly Ser Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe ThrIle Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg ValGly Tyr Ser Leu 95 100 105 Ser Gly Leu Tyr Asp Tyr Trp Gly Gln Gly ThrLeu Val Thr Val 110 115 120 Ser Ser 454 amino acids Amino Acid Linear 22Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 1 5 10 15Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 20 25 30 GluTyr Thr Met His Trp Met Lys Gln Ser His Gly Lys Ser Leu 35 40 45 Glu TrpIle Gly Gly Phe Asn Pro Lys Asn Gly Gly Ser Ser His 50 55 60 Asn Gln ArgPhe Met Asp Lys Ala Thr Leu Ala Val Asp Lys Ser 65 70 75 Thr Ser Thr AlaTyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 80 85 90 Ser Gly Ile Tyr TyrCys Ala Arg Trp Arg Gly Leu Asn Tyr Gly 95 100 105 Phe Asp Val Arg TyrPhe Asp Val Trp Gly Ala Gly Thr Thr Val 110 115 120 Thr Val Ser Ser AlaSer Thr Lys Gly Pro Ser Val Phe Pro Leu 125 130 135 Ala Pro Ser Ser LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly 140 145 150 Cys Leu Val Lys AspTyr Phe Pro Glu Pro Val Thr Val Ser Trp 155 160 165 Asn Ser Gly Ala LeuThr Ser Gly Val His Thr Phe Pro Ala Val 170 175 180 Leu Gln Ser Ser GlyLeu Tyr Ser Leu Ser Ser Val Val Thr Val 185 190 195 Pro Ser Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn 200 205 210 His Lys Pro Ser AsnThr Lys Val Asp Lys Lys Val Glu Pro Lys 215 220 225 Ser Cys Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Glu 230 235 240 Leu Leu Gly Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val GluVal His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn SerThr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 Leu His Gln Asp TrpLeu Asn Gly Lys Glu Tyr Lys Cys Lys Val 320 325 330 Ser Asn Lys Ala LeuPro Ala Pro Ile Glu Lys Thr Ile Ser Lys 335 340 345 Ala Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro 350 355 360 Ser Arg Glu Glu MetThr Lys Asn Gln Val Ser Leu Thr Cys Leu 365 370 375 Val Lys Gly Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser 380 385 390 Asn Gly Gln Pro GluAsn Asn Tyr Lys Thr Thr Pro Pro Val Leu 395 400 405 Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Lys Leu Thr Val Asp 410 415 420 Lys Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met 425 430 435 His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu 440 445 450 Ser Pro Gly Lys 469amino acids Amino Acid Linear 23 Met Gly Trp Ser Cys Ile Ile Leu Phe LeuVal Ala Thr Ala Thr 1 5 10 15 Gly Val His Ser Glu Val Gln Leu Val GluSer Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser CysAla Thr Ser Gly 35 40 45 Tyr Thr Phe Thr Glu Tyr Thr Met His Trp Met ArgGln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Gly Ile Asn Pro LysAsn Gly 65 70 75 Gly Thr Ser His Asn Gln Arg Phe Met Asp Arg Phe Thr IleSer 80 85 90 Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Gln Met Asn Ser Leu95 100 105 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Gly110 115 120 Leu Asn Tyr Gly Phe Asp Val Arg Tyr Phe Asp Val Trp Gly Gln125 130 135 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser140 145 150 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr155 160 165 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val170 175 180 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr185 190 195 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser200 205 210 Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr215 220 225 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr230 235 240 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr290 295 300 Val Asp Gly Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu305 310 315 Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val320 325 330 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val335 340 345 Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys350 355 360 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro365 370 375 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu380 385 390 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser395 400 405 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu410 415 420 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp425 430 435 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met440 445 450 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu455 460 465 Ser Pro Gly Lys 214 amino acids Amino Acid Linear 24 Asp ValGln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu 1 5 10 15 Gly AspArg Val Thr Ile Asn Cys Arg Ala Ser Gln Asp Ile Asn 20 25 30 Asn Tyr LeuAsn Trp Tyr Gln Gln Lys Pro Asn Gly Thr Val Lys 35 40 45 Leu Leu Ile TyrTyr Thr Ser Thr Leu His Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly SerGly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 65 70 75 Ser Asn Leu Asp Gln GluAsp Ile Ala Thr Tyr Phe Cys Gln Gln 80 85 90 Gly Asn Thr Leu Pro Pro ThrPhe Gly Gly Gly Thr Lys Val Glu 95 100 105 Ile Lys Arg Thr Val Ala AlaPro Ser Val Phe Ile Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys SerGly Thr Ala Ser Val Val Cys Leu 125 130 135 Leu Asn Asn Phe Tyr Pro ArgGlu Ala Lys Val Gln Trp Lys Val 140 145 150 Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln Glu Ser Val Thr Glu 155 160 165 Gln Asp Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser Thr Leu Thr 170 175 180 Leu Ser Lys Ala Asp Tyr GluLys His Lys Val Tyr Ala Cys Glu 185 190 195 Val Thr His Gln Gly Leu SerSer Pro Val Thr Lys Ser Phe Asn 200 205 210 Arg Gly Glu Cys 233 aminoacids Amino Acid Linear 25 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu ValAla Thr Ala Thr 1 5 10 15 Gly Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysArg Ala Ser 35 40 45 Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln LysPro Gly 50 55 60 Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu HisSer 65 70 75 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr80 85 90 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 95100 105 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro Thr Phe Gly Gln Gly 110115 120 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 125130 135 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 140145 150 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 155160 165 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 170175 180 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 185190 195 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 200205 210 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 215220 225 Lys Ser Phe Asn Arg Gly Glu Cys 230 122 amino acids Amino AcidLinear 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 15 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr 2025 30 Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 4045 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Thr Thr Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser 65 70 75 LysAsn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr AlaVal Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser 95 100 105 Asp TrpTyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120 Ser Ser

We claim:
 1. A humanized antibody that binds p185^(HER2) with a bindingaffinity of about 0.22 nm or stronger affinity.
 2. The humanizedantibody of claim 1 which comprises an Fc region.
 3. The humanizedantibody of claim 2 wherein the Fc region is a human γ1 constant region.4. The humanized antibody of claim 3 wherein the Fc region is a non-Aallotype human γ1 constant region.
 5. The humanized antibody of claim 1that has cytotoxic activity in the presence of human effector cells,wherein the cytotoxic activity is selective for cell types whichoverexpress p185^(HER2).
 6. The humanized antibody of claim 5 thatcomprises a non-A allotype human γ1 constant region.
 7. The humanizedantibody of claim 1 which inhibits proliferation of SKBR3 cells.
 8. Thehumanized antibody of claim 1 which mediates lysis of p185^(HER2)overexpressing cells by complement activation or antibody dependentcellular cytotoxicity (ADCC).
 9. The humanized antibody of claim 1 whichmediates lysis of p185^(HER2) overexpressing cells by complementactivation and antibody dependent cellular cytotoxicity (ADCC).
 10. Thehumanized antibody of claim 1 which is conjugated with a cytotoxicmoiety.
 11. The humanized antibody of claim 1 which is glycosylated. 12.A humanized antibody variant of a nonhuman antibody that bindsp185^(HER2), wherein the humanized antibody variant has humanreplacements of nonhuman antibody Complementarity Determining Region(CDR) amino acid residues.
 13. The humanized antibody of claim 12wherein the antibody binds p185^(HER2) with stronger affinity than thenonhuman antibody from which CDR amino acid residues of the humanizedantibody are derived.